Protegrins

ABSTRACT

Peptide-based compounds containing four invariant cysteine residues which have been optionally oxidized to contain two intramolecular disulfide bonds, or modified forms where the cysteines are replaced are useful as preservatives and in preventing, treating, or ameliorating viral or microbial infection in animals and plants, and in inactivating endotoxin. These compounds, in one embodiment, are of the formula:  
     A 1 -A 2 -A 3 -A 4 -A 5 -C 6 -A 7 -C 8 -A 9 -A 10 -A 11 -A 12 -C 13 -A 14 -C 15 -A 16 -(A 17 -A 18 )   (1)  
     and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, which is either in the optionally —SH stablizied linear or in a cystine-bridged form  
     wherein each of A 1  and A 9  is independently a basic amino acid;  
     each of A 2  and A 3  is independently a small amino acid;  
     each of A 5 , A 7 , A 12 , A 14  and A 16  is independently a hydrophobic amino acid;  
     A 4  is a basic or a small amino acid;  
     A 10  is a basic or a small amino acid or is proline;  
     A 11  is a basic or hydrophobic amino acid;  
     A 17  is not present or, if present, is a small amino acid;  
     A 18  is not present or, if present, is a basic amino acid; or a  
     modified form of formula (1) and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof wherein each of 1-4 cysteines is independently replaced by a hydrophobic amino acid or a small amino acid.

[0001] This application is a continuation of application Ser. No.08/499,523, filed 7 Jul., 1995, which is a continuation-in-part of U.S.Ser. No. 08/451,832, filed 26 May, 1995, which is a continuation-in-partof U.S. Ser. No. 08/243,879 filed 17 May 1994, which is acontinuation-in-part of U.S. Ser. No. 08/182,483 filed 13 Jan. 1994,which is a continuation-in-part of U.S. Ser. No. 08/095,769 filed 26Jul. 1993, which is a continuation-in-part of U.S. Ser. No. 08/093,926filed 20 Jul. 1993. The contents of these applications are incorporatedherein by reference.

[0002] This invention was made with funding from NIH Grant No. A122839.The U.S. Government has certain rights in this invention.

TECHNICAL FIELD

[0003] The invention relates to the field of antibiotic peptides. Inparticular, the invention concerns short peptides, some of which areisolated from porcine leukocytes, that have a wide range ofantimicrobial activities.

BACKGROUND ART

[0004] One of the defense mechanisms against infection by both animalsand plants is the production of peptides that have antimicrobial andantiviral activity. Various classes of these peptides have been isolatedfrom tissues both of plants and animals. One well known class of suchpeptides is the tachyplesins which were first isolated from thehemocytes of the horseshoe crab as described by Nakamura, T. et al. JBiol Chem (1988) 263:16709-16713. This article described the initialtachyplesin isolated, Tachyplesin I, from the Japanese species.Tachyplesin I is a 17-amino acid amidated peptide containing fourcysteine residues providing two intramolecular cystine bonds. A laterarticle by this group, Miyata, T. et al. J Biochem (1989) 106:663-668,reports the isolation of a second tachyplesin, Tachyplesin II,consisting of 17 residues amidated at the C-terminus, also containingfour cysteine residues and two intramolecular disulfide bonds. Twoadditional 18-mers, called polyphemusins, highly homologous toTachyplesin II and containing the same positions for the four cysteineresidues, were also isolated from the American horseshoe crab.Polyphemusin I and Polyphemusin II differ from each other only in thereplacement of one arginine residue by a lysine. All of the peptideswere described as having antifungal and antibacterial activity. A laterarticle by Murakami, T. et al. Chemotherapy (1991) 37:327-334, describesthe antiviral activity of the tachyplesins with respect to vesicularstomatitis virus; Herpes Simplex Virus I & II, Adenovirus I, Reovirus IIand Poliovirus I were resistant to inactivation by Tachyplesin I.Morimoto, M. et al. Chemotherapy (1991) 37:206-211, found thatTachyplesin I was inhibitory to Human Immunodeficiency Virus. Thisanti-HIV activity was found also to be possessed by a synthetic analogof Polyphemusin II as described by Nakashima, H. et al. AntimicrobialAgents and Chemotherapy (1992) 1249-1255. Antiviral peptides have alsobeen found in rabbit leukocytes as reported by Lehrer, R. I. et al. JVirol (1985) 54:467-472.

[0005] Other important classes of cysteine-containing antimicrobialpeptides include the defensins, β-defensins and insect defensins. Thedefensins are somewhat longer peptides characterized by six invariantcysteines and three intramolecular cystine disulfide bonds. Defensinswere described by Lehrer, R. I. et al. Cell (1991) 64:229-230; Lehrer,R. I. et al. Ann Rev Immunol (1993) 11:105-128. A review ofmammalian-derived defensins by Lehrer, R. I. et al. is found in AnnualReview Immunol (1993) 11:105-128; three patents have issued on thedefensins: U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; and U.S.Pat. No. 4,543,252. Defensins have been found in the polymorphonucleatedneutrophils (PMN) of humans and of several other animals, as well as inrabbit pulmonary alveolar macrophages, and in murine small intestinalepithelial (Paneth) cells and in corresponding cells in humans.

[0006] β-Defensins are found in bovine respiratory epithelial cells,bovine granulocytes and avian leukocytes. See Selsted, M. E. et al. JBiol Chem (1993) 288:6641-6648 and Diamond, G. et al. Proc Natl Acad Sci(USA) (1991) 88:3952-3958. Insect defensins have been reported byLambert, J. et al. Proc Natl Acad Sci (USA) (1989) 88:262-265.

[0007] Antifungal and antibacterial peptides and proteins have also beenfound in plants (Broekaert, W. F. et al. Biochemistry (1992)31:4308-4314) as reviewed by Cornelissen, B. J. C. et al. Plant Physiol(1993) 101:709-712. Expression systems for the production of suchpeptides have been used to transform plants to protect the plantsagainst such infection as described, for example, by Haln, R. et al.Nature (1993) 361:153-156.

[0008] The present invention provides a new class of antimicrobial andantiviral peptides, designated “protegrins” herein, representativemembers of which have been isolated from porcine leukocytes. Thesepeptides are useful as antibacterial antiviral and antifungal agents inboth plants and animals.

[0009] The isolation of the protegrin peptides of the invention wasreported by the present applicants in a paper by Kokryakov, V. N. et al.FEBS (1993) 337:231-236 (July issue). A later publication of this groupdescribed the presence of a new protegrin, whose sequence, and that ofits precursor, was deduced from its isolated cDNA clone. Zhao, C et al,FEBS Letters (1994) 346:285-288. An additional paper disclosing cationicpeptides from porcine neutrophils was published by Mirgorodskaya, O. A.et al. FEBS (1993) 330:339-342 (September issue). Storici, P. et al.Biochem Biophys Res Comm (1993) 196:1363-1367, report the recovery of aDNA sequence which encodes a pig leukocyte antimicrobial peptide with acathelin-like prosequence. The peptide is reported to be one of theprotegrins disclosed hereinbelow. Additional publications related toprotegrins are Harwig, S. S. L., et al. J. Peptide Sci. (1995) in press;and Zhao, C., et al. FEBS-MS MB-283 (1995) in press.

[0010] The protegrins of the invention have also been found to bind toendotoxins—i.e., the lipopolysaccharide (LPS) compositions derived fromgram-negative bacteria which are believed responsible for gram-negativesepsis. This type of sepsis is an extremely common condition and isoften fatal. Others have attempted to design and study proteins whichbind LPS/endotoxin, and illustrative reports of these attempts appear inRustici, A. et al. Science (1993) 259:361-364; Matsuzaki, K. et al.Biochemistry (1993) 32:11704-11710; Hoess, A. et al. EMBO J (1993)12:3351-3356; and Elsbach, P. et al. Current Opinion in Immunology(1993) 5:103-107. The protegrins of the present invention provideadditional compounds which are capable of inactivating of LPS andameliorating its effects.

[0011] In addition to the foregoing, the protegrins of the invention areeffective in inhibiting the growth of organisms that are associated withsexually transmitted diseases. It is estimated that 14 million peopleworld-wide are infected with HIV and that millions of women sustainpelvic inflammatory disease each year. Chlamydia trachomatis andNeisseria gonorrhoeae cause over half of this inflammatory diseasealthough E. coli, Mycoplasma hominis and other infectious microorganismscan also be responsible. Pathogens include viral, bacterial, fungal andprotozoan pathogens. It is especially important that the antibioticsused to combat these infections be effective under physiologicalconditions. The protegrins of the present invention offer theseproperties.

DISCLOSURE OF THE INVENTION

[0012] In one embodiment, the invention is directed to peptides of 16-18amino acid residues characterized by four invariant cysteines and eitherby a characteristic pattern of basic and hydrophobic amino acids and/orbeing isolatable from animal leukocytes using the method of theinvention. In a second embodiment, the invention is directed to theabove peptides wherein 1-4 of these cysteines is replaced by ahydrophobic or small amino acid. All of these peptides can be producedsynthetically and some can be produced recombinantly or can be isolatedfrom their native sources and purified for use as preservatives or inpharmaceutical compositions in treating or preventing infection inanimals. Alternatively, the peptides can be formulated into compositionswhich can be applied to plants to protect them against viral ormicrobial infection. In still another approach, the DNA encoding thepeptides can be expressed in situ, in animals or preferably in plants,to combat infections. The peptides are also useful as standards inantimicrobial assays and in binding endotoxins.

[0013] Accordingly, in one aspect, the invention is directed to apurified and isolated or recombinantly produced compound of the formula

A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁₀-A₁₁-A₁₂-C₁₃-A₁₄-C₁₅-A₁₆-(A₁₇-A₁₈)   (1)

[0014] and the N-terminal acylated and/or C-terminal amidated oresterified forms thereof, which is either in the optionally —SHstabilized linear or in a cystine-bridged form wherein each of A₁ and A₉is independently a basic amino acid;

[0015] each of A₂ and A₃ is independently a small amino acid;

[0016] each of A₅, A₇, A₁₂, A₁₄ and A₁₆ is independently a hydrophobicamino acid;

[0017] A₄ is a basic or a small amino acid;

[0018] A₁₀ is a basic or a small amino acid or is proline;

[0019] A₁₁ is a basic or a hydrophobic amino acid;

[0020] A₁₇ is not present or, if present, is a small amino acid;

[0021] A₁₈ is not present or, if present, is a basic amino acid, or a

[0022] modified form of formula (1) and the N-terminal acylated and/orC-terminal amidated or esterified forms thereof wherein at least one ofthe 4 cysteines is independently replaced by a hydrophobic amino acid ora small amino acid.

[0023] In still other aspects, the invention is directed to recombinantmaterials useful for the production of the peptides of the invention aswell as plants or animals modified to contain expression systems for theproduction of these peptides. The invention is also directed topharmaceutical compositions and compositions for application to plantscontaining the peptides of the invention as active ingredients orcompositions which contain expression systems for production of thepeptides or for in situ expression of the nucleotide sequence encodingthese peptides. The invention is also directed to methods to prepare theinvention peptides synthetically, to antibodies specific for thesepeptides, and to the use of the peptides as preservatives.

[0024] In other aspects, the invention is directed to the use of thecompounds of the invention as standards in antimicrobial assays. Thecompounds may also be used as antimicrobials in solutions useful in eyecare, such as contact lens solutions, and in topical or otherpharmaceutical compositions for treatment of sexually transmitteddiseases (STDs). The invention is also directed to use of the inventioncompounds as preservatives for foods or other perishables. As theinvention peptides can inactivate endotoxin, the invention is alsodirected to a method to inactivate endotoxins using the compounds of theinvention and to treat gram-negative sepsis by taking advantage of thisproperty.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows the elution pattern of a concentrate of theultrafiltrate of porcine leukocytes applied to a Biogel P10 column.

[0026]FIG. 2 shows the antibacterial activity of the P10 fractionsobtained from elution of the column described in FIG. 1.

[0027]FIG. 3 shows an elution pattern obtained when fractions 76-78 fromthe Biogel P10 column of FIG. 1 is applied to HPLC.

[0028]FIG. 4 shows the antimicrobial activity of the purified porcineprotegrins of the invention:

[0029]FIG. 4a shows antibacterial activity against E. Coli;

[0030]FIG. 4b shows antibacterial activity against Listeriamonocytogenes;

[0031]FIG. 4c shows antifungal activity against Candida albicans;

[0032]FIG. 4d shows antibacterial activity against S. aureus.

[0033]FIG. 4e shows antibacterial activity against K. pneumoneae.

[0034]FIG. 5 shows the effect of various test conditions onantimicrobial activity:

[0035]FIG. 5a shows activity against Candida albicans in 100 μM NaCl;

[0036]FIG. 5b shows activity against E. Coli in 100 μM NaCl;

[0037]FIG. 5c shows activity against Candida albicans in 90% fetal calfserum.

[0038]FIG. 6 shows the antimicrobial activity of the linear forms of theprotegrins under various test conditions:

[0039]FIG. 6a shows the activity against E. coli in 10 mMphosphate-citrate buffer, pH 6.5;

[0040]FIG. 6b shows the activity against E. coli in the same buffer with100 mM NaCl;

[0041]FIG. 6c shows the activity against L. monocytogenes in the bufferof FIGS. 6a-6 b;

[0042]FIG. 6d shows the activity against L. monocytogenes in the samebuffer with the addition of 100 mM NaCl;

[0043]FIG. 6e shows the activity against C. albicans in the presence of10 mM phosphate; and

[0044]FIG. 6f shows the activity against C. albicans in the presence of10 mM phosphate plus 100 mM NaCl.

[0045]FIG. 7 shows a composite of cDNA encoding the precursors of PG-1,PG-2, PG-3 and PG-4.

[0046]FIG. 8 shows the nucleotide sequence and the deduced amino acidsequence of the genomic DNA encoding the precursor protein for theantimicrobial compounds of the invention PG-1, PG-3, and PG-5.

[0047]FIG. 9 shows the organization of the protegrin genomic DNA.

[0048]FIG. 10 shows the amino acid sequences of the protegrins PG-1 toPG-5.

[0049]FIGS. 11a-11 c show the antimicrobial activity of syntheticallyprepared PG-5 as compared to that of synthetically prepared PG-1.

[0050]FIGS. 12a-12 d show the effects of various protegrins againstvarious target microbes.

[0051]FIG. 13 shows a graphical representation of the effects of thekite and bullet forms of PG-1 against gram positive bacteria.

[0052]FIG. 14 shows a graphical representation of the effects of thekite and bullet forms of PG-1 against gram negative bacteria.

[0053]FIG. 15 is a graphical representation of the antimicrobialactivity of the snake form of PG-1 against gram positive bacteria.

[0054]FIG. 16 is a graphical representation of the antimicrobialactivity of the snake form of PG-1 against gram negative bacteria.

MODES OF CARRYING OUT THE INVENTION

[0055] The peptides of the invention are described by the formula:

A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁₀-A₁₁-A₁₂-C₁₃-A₁₄-C₁₅-A₁₆-(A₁₇-A₁₈),   (1)

[0056] and its defined modified forms. Those peptides which occur innature must be in purified and isolated form or prepared recombinantly.

[0057] The designation A_(n) in each case represents an amino acid atthe specified position in the peptide. As A₁₇ and A₁₈ may or may not bepresent, the peptides of the invention contain either 16, 17 or 18 aminoacids. The positions of the cysteine residues, shown as C in Formula(1), are invariant in the peptides of the invention; however, in themodified forms of the peptides of Formula (1), also included within thescope of the invention, at least one of 1-4 of these cysteines may bereplaced by a hydrophobic or small amino acid.

[0058] The amino terminus of the peptide may be in the free amino formor may be acylated by a group of the formula RCO—, wherein R representsa hydrocarbyl group of 1-6C. The hydrocarbyl group is saturated orunsaturated and is typically, for example, methyl, ethyl, i-propyl,t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl,hexyne-4-yl, and the like.

[0059] The C-terminus of the peptides of the invention may be in theform of the underivatized carboxyl group, either as the free acid or anacceptable salt, such as the potassium, sodium, calcium, magnesium, orother salt of an inorganic ion or of an organic ion such as caffeine.The carboxyl terminus may also be derivatized by formation of an esterwith an alcohol of the formula ROH, or may be amidated by an amine ofthe formula NH₃, or RNH₂, or R₂NH, wherein each R is independentlyhydrocarbyl of 1-6C as defined above. Amidated forms of the peptideswherein the C-terminus has the formula CONH₂ are preferred.

[0060] As the peptides of the invention contain substantial numbers ofbasic amino acids, the peptides of the invention may be supplied in theform of the acid addition salts. Typical acid addition salts includethose of inorganic ions such as chloride, bromide, iodide, fluoride orthe like, sulfate, nitrate, or phosphate, or may be salts of organicanions such as acetate, formate, benzoate and the like. Theacceptability of each of such salts is dependent on the intended use, asis commonly understood.

[0061] The peptides of the invention that contain at least two cysteinesmay be in straight-chain or cyclic form. The straight-chain forms areconvertible to the cyclic forms, and vice versa. Methods for formingdisulfide bonds to create the cyclic peptides are well known in the art,as are methods to reduce disulfides to form the linear compounds. Thelinear compounds can be stabilized by addition of a suitable alkylatingagent such as iodoacetamide.

[0062] The cyclic forms are the result of the formation of cystinelinkages among all or some of the four invariant cysteine residues.Cyclic forms of the invention include all possible permutations ofcystine bond formation; if the cysteines are numbered in order of theiroccurrence starting at the N-terminus as C₆, C₈, C₁₃ and C₁₅, thesepermutations include:

[0063] C₆-C₈;

[0064] C₆-C₁₃;

[0065] C₆-C₁₅;

[0066] C₈-C₁₃;

[0067] C₈-C₁₅;

[0068] C₁₃-C₁₅;

[0069] C₆-C₈, C₁₃-C₁₅;

[0070] C₆-C₁₃, C₈-C₁₅; and

[0071] C₆-C₁₅, C₈-C₁₃.

[0072] In the modified forms of the peptides, where 1-4 cysteines arereplaced, similar permutations are available when 2-3 cysteines arepresent.

[0073] The native forms of the protegrins contain two cystine bonds: onebetween the cysteine at position 6 and the cysteine at position 15 andthe other between the cysteine at position 8 and the cysteine atposition 13. Accordingly, in those embodiments having two cystinelinkages, the C₆-C₁₅, C₈-C₁₃ form is preferred. However, it has beenfound by the present applicants that forms of the protegrins containingonly one cystine linkage are active and easily prepared. Preferred amongembodiments having only one cystine linkage are those represented byC₆-C₁₅ alone and by C₈-C₁₃ alone.

[0074] Forms containing a C₆-C₁₅ cystine as the only cystine linkage aregenerally designated “bullet” forms of the protegrins; those wherein thesole cystine is C₈-C₁₃ are designated the “kite” forms. The bullet andkite forms can most conveniently be made by replacing the cystines atthe positions not to be linked by cystine with a neutral amino acid,preferably a small amino acid such as glycine, serine, alanine orthreonine and less preferably a neutral polar amino acid such asasparagine or glutamine or by a hydrophobic amino acid. Thus, in someembodiments of the bullet form, each of C₈ and C₁₃ is independentlyalanine, serine, threonine or glycine, preferably both are alanine.Conversely, in the kite form C₆ and C₁₅ are thus replaced.

[0075] As the linearalized forms of the native cyclic peptides havevaluable activities, even when chemically stabilized to preserve thesulfhydryl form of cysteine for example, by reaction with iodoacetamide,the compounds of the invention also include linearalized forms which arestabilized with suitable reagents. As defined herein, “SH-stabilized”forms of the peptides of the invention contain sulfhydryl groups reactedwith standard reagents to prevent reformation into disulfide linkages.

[0076] An alternative approach to providing linear forms of theprotegrins of the invention comprises use of the modified form of thepeptides where cysteine residues are replaced by amino acids which donot form cystine linkages. In this instance, too, all 4 (or at least 3)of the cystines at positions 6, 8, 13, and 15 are replaced by polarneutral or small amino acids as listed above. It is preferred that all 4cysteine residues be replaced in order to minimize the likelihood ofintermolecular bonding.

[0077] The amino acids denoted by A_(n) may be those encoded by the geneor analogs thereof, and may also be the D-isomers thereof. One preferredembodiment of the peptides of the invention is that form wherein all ofthe residues are in the D-configuration thus conferring resistance toprotease activity while retaining antimicrobial or antiviral properties.The resulting protegrins are themselves enantiomers of the nativeL-amino acid-containing forms.

[0078] The amino acid notations used herein are conventional and are asfollows: One-Letter Three-Letter Amino Acid Symbol Symbol Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

[0079] The amino acids not encoded genetically are abbreviated asindicated in the discussion below.

[0080] In the specific peptides shown in the present application, theL-form of any amino acid residue having an optical isomer is intendedunless the D-form is expressly indicated by a dagger superscript (†).

[0081] The compounds of the invention are peptides which are partiallydefined in terms of amino acid residues of designated classes. Aminoacid residues can be generally subclassified into major subclasses asfollows:

[0082] Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

[0083] Basic: The residue has a positive charge due to association withH ion at physiological pH and the residue is attracted by aqueoussolution so as to seek the surface positions in the conformation of apeptide in which it is contained when the peptide is in aqueous mediumat physiological pH.

[0084] Hydrophobic: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

[0085] Neutral/polar: The residues are not charged at physiological pH,but the residue is not sufficiently repelled by aqueous solutions sothat it would seek inner positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium.

[0086] This description also characterizes certain amino acids as“small” since their side chains are not sufficiently large, even ifpolar groups are lacking, to confer hydrophobicity. “Small” amino acidsare those with four carbons or less when at least one polar group is onthe side chain and three carbons or less when not.

[0087] It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged,” a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

[0088] Amino acid residues can be further subclassified as cyclic ornoncyclic, and aromatic or nonaromatic, self-explanatory classificationswith respect to the side-chain substituent groups of the residues, andas small or large. The residue is considered small if it contains atotal of four carbon atoms or less, inclusive of the carboxyl carbon,provided an additional polar substituent is present; three or less ifnot. Small residues are, of course, always nonaromatic.

[0089] For the naturally occurring protein amino acids,subclassification according to the foregoing scheme is as follows.

[0090] Acidic: Aspartic acid and Glutamic acid;

[0091] Basic: Noncyclic: Arginine, Lysine; Cyclic: Histidine;

[0092] Small: Glycine, Serine, Alanine, Threonine;

[0093] Polar/large: Asparagine, Glutamine;

[0094] Hydrophobic: Tyrosine, Valine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan.

[0095] The gene-encoded secondary amino acid proline is a special casedue to its known effects on the secondary conformation of peptidechains, and is not, therefore, included in a group. Cysteine residuesare also not included in these classifications since their capacity toform disulfide bonds to provide secondary structure is critical in thecompounds of the present invention.

[0096] Certain commonly encountered amino acids, which are not encodedby the genetic code, include, for example, beta-alanine (beta-Ala), orother omega-amino acids, such as 3-aminopropionic, 2,3-diaminopropionic(2,3-diaP), 4-aminobutyric and so forth, alpha-aminisobutyric acid(Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit),t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine(N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine(Nle), 2-naphthylalanine (2-Nal);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); methionine sulfoxide (MSO); and homoarginine(Har). These also fall conveniently into particular categories.

[0097] Based on the above definitions,

[0098] Sar, beta-Ala, 2,3-diaP and Aib are small;

[0099] t-BuA, t-BuG, N-MeIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic arehydrophobic;

[0100] Orn and Har are basic;

[0101] Cit, Acetyl Lys, and MSO are neutral/polar.

[0102] The various omega-amino acids are classified according to size assmall (beta-Ala and 3-aminopropionic) or as large and hydrophobic (allothers).

[0103] Other amino acid substitutions of those encoded in the gene canalso be included in peptide compounds within the scope of the inventionand can be classified within this general scheme according to theirstructure.

[0104] In all of the peptides of the invention, one or more amidelinkages (—CO—NH—) may optionally be replaced with another linkage whichis an isostere such as —CH₂NH—, —CH₂S—, —CH₂CH₂, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be madeby methods known in the art. The following references describepreparation of peptide analogs which include these alternative-linkingmoieties: Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,“Peptide Backbone Modifications” (general review); Spatola, A. F., in“Chemistry and Biochemistry of Amino Acids Peptides and Proteins,” B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (generalreview); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (generalreview); Hudson, D., et al., Int J Pest Prot Res (1979) 14:177-185(—CH₂NH—, —CH₂CH₂—); Spatola, A. F., et al., Life Sci (1986)38:1243-1249 (—CH₂—S); Hann, M. M., J Chem Soc Perkin Trans I (1982)307-314 (—CH—CH—, cis and trans); Almquist, R. G., et al., J Med Chem(1980) 23:1392-1398 (—COCH₂—); Jennings-White, C., et al., TetrahedronLett (1982) 23:2533 (—COCH₂—); Szelke, M., et al., European ApplicationEP 45665 (1982) CA:97:39405 (1982) (—CH(OH)CH₂—); Holladay, M. W., etal., Tetrahedron Lett (1983) 24:4401-4404 (—C(OH)CH₂—); and Hruby, V.J., Life Sci (1982) 31:189-199 (—CH₂—S—).

[0105] The compounds of Formula (1) are generally defined as

A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁₀-A₁₁-A₁₂-C₁₃-A₁₄-C₁₅-A₁₆-(A₁₇-A₁₈)   (1)

[0106] and the N-terminal acylated and/or C-terminal amidated oresterified forms thereof, which is either in the optionally —SHstabilized linear or in a cystine-bridged form

[0107] wherein each of A₁ and A₉ is independently a basic amino acid;

[0108] each of A₂ and A₃ is independently a small amino acid;

[0109] each of A₅, A₇, A₁₂, A₁₄ and A₁₆ is independently a hydrophobicamino acid;

[0110] A₄ is a basic or a small amino acid;

[0111] A₁₀ is a basic or a small amino acid or is proline;

[0112] A₁₁ is a basic or a hydrophobic amino acid;

[0113] A₁₇ is not present or, if present, is a small amino acid;

[0114] A₁₈ is not present or, if present, is a basic amino acid, or a

[0115] modified form of formula (1) and the N-terminal acylated and/orC-terminal amidated or esterified forms thereof wherein at least one ofthe 4 and up to all 4 cysteines is each independently replaced by ahydrophobic amino acid or a small amino acid.

[0116] In preferred embodiments of the compounds of the invention, eachof A₁ and A₉ is independently selected from the group consisting of R, Kand Har; more preferably, both A₁ and A₉ are R.

[0117] In another class of preferred embodiments, each of A₂ and A₃ isindependently selected from the group consisting of G, A, S and T; morepreferably, A₂ and A₃ are G.

[0118] In another set of preferred embodiments, A₄ is selected from thegroup consisting of R, K, Har, G, A, S and T; more preferably, A₄ is Ror G.

[0119] In another set of preferred embodiments, each of A₅, A₁₄ and A₁₆is selected independently from the group consisting of I, V, L, Nle andF; preferably I, V, L and F.

[0120] In another set of preferred embodiments, each of A₇ and A₁₂ isindependently selected from the group consisting of I, V, L, W, Y and F;preferably A₇ is Y and A₁₂ is I or F.

[0121] In another set of preferred embodiments, A₁₀ is R, G or P.

[0122] In another set of preferred embodiments, A₁₁ is R or W.

[0123] A₁₇, when present, is preferably G, A, S or T, most preferably G;

[0124] A₁₈, when present, is preferably R, K or Har, most preferably R.

[0125] As described above, the compounds of Formula (1) are either incyclic or noncyclic (linearalized) form or may be modified wherein 1-4of the cysteines is replaced by a small amino acid residue or ahydrophobic residue or a polar large amino acid residue. If thelinearalized forms of the compound of Formula (1) are prepared, or iflinearalized forms of those modified peptides which contain at least twocysteines are prepared, it is preferred that the sulfhydryl groups bestabilized by addition of a suitable reagent. Preferred embodiments forthe hydrophobic amino acid to replace cysteine residues are I, V, L andNLe, preferably I, V or L. Preferred small amino acids to replace thecysteine residues include G, A, S and T, most preferably G. Preferredlarge polar amino acids are N and Q.

[0126] In an alternative embodiment, the peptides of the invention aredefined as described by Formula (1), but wherein the definitions ofA_(n) in each case are determined by the isolatability of the peptidefrom animal leukocytes by the invention method. The invention methodcomprises the steps of providing an ultrafiltrate of a lysate of animalleukocytes and isolating peptides of 16-18 amino acids. These peptidescan further be defined by the ability of DNA encoding them to hybridizeunder stringent conditions to DNA encoding the peptides exemplified asPG-1, PG-2, PG-3, PG-4 and PG-5 herein.

[0127] Particularly preferred compounds of the invention are: Unmodifiedforms PG-1: R-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-V-G-R PG-2:R-G-G-R-L-C-Y-C-R-R-R-F-C-I-C-V PG-3:R-G-G-G-L-C-Y-C-R-R-R-F-C-V-C-V-G-R PG-4:R-G-G-R-L-C-Y-C-R-G-W-I-C-F-C-V-G-R PG-5:R-G-G-R-L-C-Y-C-R-P-R-F-C-V-C-V-G-R R-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-VK-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-V R-G-G-Har-L-C-Y-C-R-R-R-F-C-V-C-VR-G-G-Har-L-C-Y-C-Har-R-R-F-C-V-C-V-G-RR-G-G-R-V-C-Y-C-R-Har-R-F-C-V-C-V-G-RR-G-G-R-L-C-Y-C-R-K-K-W-C-V-C-V-G-RR-G-G-R-L-C-Y-C-R-Har-R-Y-C-V-C-V-G-RR-G-S-G-L-C-Y-C-R-R-K-W-C-V-C-V-G-R R-A-T-R-I-C-F-C-R-R-R-F-C-V-C-V-G-RR-G-G-K-V-C-Y-C-R-Har-R-F-C-V-C-V-G-RR-A-T-R-I-C-F-C-R′-R-R-F-C-V-C-V-G-RtR-G-G-K-V-C-Y-C-R-Har′-R-F-C-V-C-V-G-R PG-1:R-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-V-G-R (all ′) PG-2:R-G-G-R-L-C-Y-C-R-R-R-F-C-I-C-V (all ′) PG-3:R-G-G-G-L-C-Y-C-R-R-R-F-C-V-C-V-G-R (all ′) PG-4:R-G-G-R-L-C-Y-C-R-G-W-I-C-F-C-V-G-R (all ′) PG-5:R-G-G-R-L-C-Y-C-R-P-R-F-C-V-C-V-G-R

[0128] both the linear and mono- and bicyclic forms thereof, andincluding the N-terminal acylated and C-terminal amidated forms;Modified forms R-G-G-R-L-V-Y-C-R-R-R-F-C-V-C-V-G-RR-G-G-R-L-G-Y-C-R-R-R-F-C-I-C-V R-G-G-G-L-C-Y-G-R-R-R-F-C-V-C-V-G-RR-G-G-R-L-G-Y-G-R-R-R-F-G-V-C-V K-G-G-R-L-V-Y-V-R-R-R-F-I-V-C-VR-G-G-Har-L-C-Y-C-R-R-R-F-C-V-G-VR-G-G-Har-L-C-Y-C-Har-R-R-F-C-V-L-V-G-RR-G-G-R-V-C-Y-V-R-Har-R-F-L-V-G-V-G-RR-G-G-R-L-C-Y-S-R-K-K-W-C-V-S-V-G-RR-G-G-R-L-C-Y-C-R-Har-R-Y-S-V-V-V-G-RR-G-S-G-L-S-Y-C-R-R-K-W-G-V-C-V-G-R R-A-T-R-I-S-F-S-R-R-R-F-S-V-S-V-G-RR-G-G-K-V-C-Y-G-R-Har-R-F-S-V-C-V-G-RR-A-T-R-I-V-F-C-R′-R-R-F-G-V-C-V-G-R′R-G-G-K-V-C-Y-L-R-Har′-R-F-L-V-C-V-G-RR-G-G-R-I-C-F-L-R-P-R-I-G-V-C-V-G-R

[0129] both the linear and cyclic (where possible) forms thereof, andincluding the N-terminal acylated and C-terminal amidated forms.

[0130] Particularly preferred are compounds wherein a single cystinebond is formed between C6 and C15 or between C8 and C13 wherein fourcompounds having a cystine bond between C8 and C13 each of C6 and C15 isindependently replaced by “X” wherein X is a hydrophobic, a small, or alarge polar amino acid. Similarly, where the single cystine bond isbetween C8 and C13, each of C6 and C15 is independently replaced by X asdefined above. Also preferred are the “snake” forms of the compounds ofthe invention where all 4 cysteines are replaced by X as defined above.Particularly preferred embodiments of these compounds of the inventioninclude:

Snake form-1 R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-V-G-R Snake form-2R-G-G-R-L-X-Y-X-R-R-R-F-X-I-X-V Snake form-3R-G-G-G-L-X-Y-X-R-R-R-F-X-V-X-V-G-R Snake form-4R-G-G-R-X-L-X-Y-R-G-W-I-X-F-X-V-G-R Snake form-5R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-V-G-R

[0131] wherein X is as defined above.

[0132] Particularly preferred embodiments of X are those wherein X is asmall amino acid, especially S and A, especially A.

[0133] Preparation of the Invention Compounds

[0134] The invention compounds, often designated herein “protegrins” areessentially peptide backbones which may be modified at the N— orC-terminus and also may contain one or two cystine disulfide linkages.The peptides may first be synthesized in noncyclized form. Thesepeptides may then be converted to the cyclic peptides if desired bystandard methods of cystine bond formation. As applied to the protegrinsherein, “cyclic forms” refers to those forms which contain cyclicportions by virtue of the formation of disulfide linkages betweencysteine residues in the peptide. If the straight-chain forms arepreferred, it is preferable to stabilize the sulfhydryl groups for anypeptides of the invention which contain two or more cysteine residues.

[0135] Standard methods of synthesis of peptides the size of protegrinsare known. Most commonly used currently are solid phase synthesistechniques; indeed, automated equipment for systematically constructingpeptide chains can be purchased. Solution phase synthesis can also beused but is considerably less convenient. When synthesized using thesestandard techniques, amino acids not encoded by the gene andD-enantiomers can be employed in the synthesis. Thus, one very practicalway to obtain the compounds of the invention is to employ these standardchemical synthesis techniques.

[0136] In addition to providing the peptide backbone, the N— and/orC-terminus can be derivatized, again using conventional chemicaltechniques. The compounds of the invention may optionally contain anacyl group, preferably an acetyl group at the amino terminus. Methodsfor acetylating or, more generally, acylating, the free amino group atthe N-terminus are generally known in the art; in addition, theN-terminal amino acid may be supplied in the synthesis in acylated form.

[0137] At the carboxy terminus, the carboxyl group may, of course, bepresent in the form of a salt; in the case of pharmaceuticalcompositions this will be a pharmaceutically acceptable salt. Suitablesalts include those formed with inorganic ions such as NH₄ ⁺, Na⁺, K⁺,Mg⁺⁺, Ca⁺⁺, and the like as well as salts formed with organic cationssuch as those of caffeine and other highly substituted amines. Thecarboxy terminus may also be esterified using alcohols of the formulaROH wherein R is hydrocarbyl (1-6C) as defined above. Similarly, thecarboxy terminus may be amidated so as to have the formula —CONH₂,—CONHR, or —CONR₂, wherein each R is independently hydrocarbyl (1-6C) asherein defined. Techniques for esterification and amidation as well asneutralizing in the presence of base to form salts are all standardorganic chemical techniques.

[0138] If the peptides of the invention are prepared under physiologicalconditions, the side-chain amino groups of the basic amino acids will bein the form of the relevant acid addition salts.

[0139] Formation of disulfide linkages, if desired, is conducted in thepresence of mild oxidizing agents. Chemical oxidizing agents may beused, or the compounds may simply be exposed to the oxygen of the air toeffect these linkages. Various methods are known in the art. Processesuseful for disulfide bond formation have been described by Tam, J. P. etal., Synthesis (1979) 955-957; Stewart, J. M. et al, “Solid PhasePeptide Synthesis” 2d Ed. Pierce Chemical Company Rockford, Ill. (1984);Ahmed A. K. et al., J Biol Chem (1975) 250:8477-8482 and Pennington M.W. et al., Peptides 1990, E. Giralt et al., ESCOM Leiden, TheNetherlands (1991) 164-166. An additional alternative is described byKamber, B. et al., Helv Chim Acta (1980) 63:899-915. A method conductedon solid supports is described by Albericio Int J Pept Protein Res(1985) 26:92-97.

[0140] A particularly preferred method is solution oxidation usingmolecular oxygen. This method has been used by the inventors herein torefold synthetic PG-1, PG-3 in its amide or acid forms, enantioPG-1 andthe two unisulfide PG-1 compounds (C₆-C₁₅ and C₈-C₁₃). Recoveries are ashigh as 30%.

[0141] If the peptide backbone is comprised entirely of gene-encodedamino acids, or if some portion of it is so composed, the peptide or therelevant portion may also be synthesized using recombinant DNAtechniques. The DNA encoding the peptides of the invention may itself besynthesized using commercially available equipment; codon choice can beintegrated into the synthesis depending on the nature of the host.Alternatively, although less convenient, the DNA can be obtained, atleast initially, by screening a cDNA library prepared from porcineleukocytes using probes or PCR primers based on the sequences of theprotegrins described herein. This results in recovery of the naturallyoccurring sequence encoding the protegrins of the invention. Obtentionof this native sequence is significant for purposes other than thesynthesis of the protegrins per se; the availability of the naturallyoccurring sequences provides a useful probe to obtain corresponding DNAencoding protegrins of other species. Thus, cDNA libraries, for example,of leukocytes derived from other animals can be screened using thenative DNA, preferably under conditions of high stringency. Highstringency is as defined by Maniatis, et al. Molecular Cloning: aLaboratory Manual 2nd Ed, Cold Spring Harbor Laboratory Press (1989),the relevant portions of which are incorporated herein by reference.This procedure also permits recovery of allelic variants of thesepeptides from the same species.

[0142] Alternatively, the protegrins can be prepared by isolation fromleukocytes of a desired species using techniques similar to thosedisclosed herein for the isolation of porcine protegrins. In general,these techniques involve preparing a lysate of a leukocyte preparation,ultrafiltering the supernatant of the clarified lysate and recoveringthe ultrafiltrate. The ultrafiltrate is then subjected tochromatographic separation. The location of fragments havingantimicrobial and antiviral activity corresponding to protegrins can beassessed using criteria of molecular weight and assaying the fractionsfor the desired activities as described herein. The native forms ofthese peptides are believed to be the cyclic forms; if desired, thelinearalized forms can be prepared by treating the peptides withreducing agents and stabilizing the sulfhydryl groups that result.

[0143] Isolated and recombinantly produced forms of the protegrins mayrequire subsequent derivatization to modify the N— and/or C-terminusand, depending on the isolation procedure, to effect the formation ofcystine bonds as described hereinabove. Depending on the host organismused for recombinant production and the animal source from which theprotein is isolated, some or all of these conversions may already havebeen effected.

[0144] For recombinant production, the DNA encoding the protegrins ofthe invention is included in an expression system which places thesecoding sequences under control of a suitable promoter and other controlsequences compatible with an intended host cell. Types of host cellsavailable span almost the entire range of the plant and animal kingdoms.Thus, the protegrins of the invention could be produced in bacteria oryeast (to the extent that they can be produced in a nontoxic orrefractile form or utilize resistant strains) as well as in animalcells, insect cells and plant cells. Indeed, modified plant cells can beused to regenerate plants containing the relevant expression systems sothat the resulting transgenic plant is capable of self protectionvis-à-vis these infective agents.

[0145] The protegrins of the invention can be produced in a form thatwill result in their secretion from the host cell by fusing to the DNAencoding the protegrin, a DNA encoding a suitable signal peptide, or maybe produced intracellularly. They may also be produced as fusionproteins with additional amino acid sequence which may or may not needto be subsequently removed prior to the use of these compounds asantimicrobials or antivirals.

[0146] Thus, the protegrins of the invention can be produced in avariety of modalities including chemical synthesis, recombinantproduction, isolation from natural sources, or some combination of thesetechniques.

[0147] Those members of the protegrin class which occur naturally aresupplied in purified and isolated form. By “purified and isolated” ismeant free from the environment in which the peptide normally occurs (inthe case of such naturally occurring peptides) and in a form where itcan be used practically. Thus, “purified and isolated” form means thatthe peptide is substantially pure, i.e., more than 90% pure, preferablymore than 95% pure and more preferably more than 99% pure or is in acompletely different context such as that of a pharmaceuticalpreparation.

[0148] Antibodies

[0149] Antibodies to the protegrins of the invention may also beproduced using standard immunological techniques for production ofpolyclonal antisera and, if desired, immortalizing theantibody-producing cells of the immunized host for sources of monoclonalantibody production. Techniques for producing antibodies to anysubstance of interest are well known. It may be necessary to enhance theimmunogenicity of the substance, particularly as here, where thematerial is only a short peptide, by coupling the hapten to a carrier.Suitable carriers for this purpose include substances which do notthemselves produce an immune response in the mammal to be administeredthe hapten-carrier conjugate. Common carriers used include keyholelimpet hemocyanin (KLH), diphtheria toxoid, serum albumin, and the viralcoat protein of rotavirus, VP6. Coupling of the hapten to the carrier iseffected by standard techniques such as contacting the carrier with thepeptide in the presence of a dehydrating agent such asdicyclohexylcarbodiimide or through the use of linkers such as thoseavailable through Pierce Chemical Company, Chicago, Ill.

[0150] The protegrins of the invention in immunogenic form are theninjected into a suitable mammalian host and antibody titers in the serumare monitored. It should be noted, however, that some forms of theprotegrins require modification before they are able to raiseantibodies, due to their resistance to antigen processing. For example,the native form of PG-1, containing two cystine bridges isnonimmunogenic when administered without coupling to a larger carrierand was a poor immunogen even in the presence of potent adjuvants andwhen coupled through glutaraldehyde or to KLH. Applicants believe thisto be due to its resistance to attack by leukocyte serine proteases(human PMN elastase and cathepsin G) as well as to attack by an asparticprotease (pepsin) that resembles several macrophage cathepsins. The lackof immunogenicity may therefore result from resistance to processing toa linear form that can fit in the antigen-presenting pocket of thepresenting cell. Immunogenecity of these forms of the protegrins can beenhanced by cleaving the disulfide bonds.

[0151] Polyclonal antisera may be harvested when titers are sufficientlyhigh. Alternatively, antibody-producing cells of the host such as spleencells or peripheral blood lymphocytes may be harvested and immortalized.The immortalized cells are then cloned as individual colonies andscreened for the production of the desired monoclonal antibodies.

[0152] The antibodies of the invention are, of course, useful inimmunoassays for determining the amount or presence of the protegrins.Such assays are essential in quality controlled production ofcompositions containing the protegrins of the invention. In addition,the antibodies can be used to assess the efficacy of recombinantproduction of the protegrins, as well as screening expression librariesfor the presence of protegrin encoding genes.

[0153] Compositions Containing the Protegrins and Methods of Use

[0154] The protegrins of the invention are effective in inactivating awide range of microbial and viral targets, including gram-positive andgram-negative bacteria, yeast, protozoa and certain strains of virus.Accordingly, they can be used in disinfectant compositions and aspreservatives for materials such as foodstuffs, cosmetics, medicaments,or other materials containing nutrients for organisms. For use in suchcontexts, the protegrins are supplied either as a single protegrin, inadmixture with several other protegrins, or in admixture with additionalantimicrobial agents. In general, as these are preservatives in thiscontext, they are usually present in relatively low amounts, of lessthan 5%, by weight of the total composition, more preferably less than1%, still more preferably less than 0.1%.

[0155] The peptides of the invention are also useful as standards inantimicrobial assays and in assays for determination of capability oftest compounds to bind to endotoxins such as lipopolysaccharides.

[0156] For use as antimicrobials or antivirals for treatment of animalsubjects, the protegrins of the invention can be formulated aspharmaceutical or veterinary compositions. Depending on the subject tobe treated, the mode of administration, and the type of treatmentdesired—e.g., prevention, prophylaxis, therapy; the protegrins areformulated in ways consonant with these parameters. A summary of suchtechniques is found in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Co., Easton, Pa.

[0157] The protegrins are particularly attractive as an activeingredients pharmaceutical compositions useful in treatment of sexuallytransmitted diseases, including those caused by Chlamydia trachomatis,Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, Herpessimplex type 2 and HIV. Topical formulations are preferred and includecreams, salves, oils, powders, gels and the like. Suitable topicalexcipient are well known in the art and can be adapted for particularuses by those of ordinary skill.

[0158] In general, for use in treatment or prophylaxis of STDs, theprotegrins of the invention may be used alone or in combination withother antibiotics such as erythromycin, tetracycline, macrolides, forexample azithromycin and the cephalosporins. Depending on the mode ofadministration, the protegrins will be formulated into suitablecompositions to permit facile delivery to the affected areas. Theprotegrins may be used in forms containing one or two disulfide bridgesor may be in linear form. In addition, use of the enantiomeric formscontaining all D-amino acids may confer advantages such as resistance tothose proteases, such as trypsin and chymotrypsin, to which theprotegrins containing L-amino acids are less resistant.

[0159] The protegrins of the invention can be administered singly or asmixtures of several protegrins or in combination with otherpharmaceutically active components. The formulations may be prepared ina manner suitable for systemic administration or topical or localadministration. Systemic formulations include those designed forinjection (e.g., intramuscular, intravenous or subcutaneous injection)or may be prepared for transdermal, transmucosal, or oraladministration. The formulation will generally include a diluent as wellas, in some cases, adjuvants, buffers, preservatives and the like. Theprotegrins can be administered also in liposomal compositions or asmicroemulsions.

[0160] If administration is to be oral, the protegrins of the inventionmust be protected from degradation in the stomach using a suitableenteric coating. This may be avoided to some extent by utilizing aminoacids in the D-configuration, thus providing resistance to protease.However, the peptide is still susceptible to hydrolysis due to theacidic conditions of the stomach; thus, some degree of enteric coatingmay still be required.

[0161] As described in the examples below, the peptides of the inventionretain their activity against microbes in the context of boratesolutions that are commonly used in eye care products. It has also beenshown that when tested for antimicrobial activity against E. coli in thepresence and absence of lysozyme in borate buffered saline, that thepresence of lysozyme enhanced the effectiveness of PG-3. This effect wasmore pronounced when the PG-3 was autoclaved and similar patterns wereobtained for both the free-acid form and the amide. Accordingly, theprotegrins may be used as preservatives in such compositions or asantimicrobials for treatment of eye infections.

[0162] It is particularly important that the protegrins retain theiractivity under physiological conditions including relatively high salineand in the presence of serum. In addition, the protegrins are notcytotoxic with respect to the cells of higher organisms. Theseproperties, described herein below in the Examples, make themparticularly suitable for in vivo and therapeutic use.

[0163] The protegrins of the invention may also be applied to plants orto their environment to prevent viral- and microbial-induced diseases inthese plants. Suitable compositions for this use will typically containa diluent as well as a spreading agent or other ancillary agreementsbeneficial to the plant or to the environment.

[0164] Thus, the protegrins of the invention may be used in any contextwherein an antimicrobial and/or antiviral action is required. This usemay be an entirely in vitro use, or the peptides may be administered toorganisms.

[0165] In addition, the antimicrobial or antiviral activity may begenerated in situ by administering an expression system suitable for theproduction of the protegrins of the invention. Such expression systemscan be supplied to plant and animal subjects using known techniques. Forexample, in animals, pox-based expression vectors can be used togenerate the peptides in situ. Similarly, plant cells can be transformedwith expression vectors and then regenerated into whole plants which arecapable of their own production of the peptides.

[0166] A particularly useful property of the protegrins is theiractivity in the presence of serum. Unlike defensins, protegrins arecapable of exerting their antimicrobial effects in the presence ofserum.

[0167] As shown hereinbelow, the protegrins are capable of inactivatingendotoxins derived from gram-negative bacteria—i.e., lipopolysaccharides(LPS)—in standard assays. Accordingly, the protegrins may be used underany circumstances where inactivation of LPS is desired. One suchsituation is in the treatment or amelioration of gram-negative sepsis.

[0168] The protegrins of the invention, therefore, represent apeculiarly useful class of compounds because of the followingproperties:

[0169] 1) they have an antimicrobial effect with respect to a broadspectrum of target microbial systems, including viruses, includingretroviruses, bacteria, fungi, yeast and protozoa.

[0170] 2) Their antimicrobial activity is effective under physiologicalconditions—i.e., physiological saline and in the presence of serum.

[0171] 3) They are not toxic to the cells of higher organisms.

[0172] 4) They can be prepared in nonimmunogenic form thus extending thenumber of species to which they can be administered.

[0173] 5) They can be prepared in forms which are resistant to certainproteases suggesting they are antimicrobial even in lysosomes.

[0174] 6) They can be prepared in forms that resist degradation whenautoclaved, thus simplifying their preparation as components ofpharmaceuticals.

[0175] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 Isolation of PG-1, PG-2 and PG-3

[0176] Fresh porcine blood was collected into 15-liter vesselscontaining 5% EDTA in normal saline, pH 7.4 as an anticoagulant (33ml/liter blood). The blood cells were allowed to sediment for 90 minutesat room temperature and the leukocyte-rich supernatant was removed andcentrifuged at 200×g for 5.7 minutes. The pellets were pooled andsuspended in 0.84% ammonium chloride to lyse erythrocytes and theresulting leukocytes (70-75% PMN, 5-10% eosinophils, 15-25% lymphocytesand monocytes) were washed in normal saline, resuspended in ice-cold 10%acetic acid at 10⁸/ml, homogenized and stirred overnight at 4° C. Thepreparation was centrifuged at 25,000×g for 3 hours at 4° C. and thesupernatant was lyophilized and weighed.

[0177] 950 mg (dry weight) of lyophilized extract, which contained 520mg protein by BCA analysis, was stirred overnight at 4° C. in 100 ml of10% acetic acid and then centrifuged at 25,000×g for 2 hours. Thesupernate was removed and passed by pressure through a 50 ml stirredultracentrifugation cell (Amicon, Danvers Mass.) that contained a YM-5filter. The ultrafiltrate (24.5 mg protein by BCA) was concentrated to 3ml by vacuum centrifugation (SpeedVac Concentrator, Savant Instruments,Hicksville, N.Y.), applied to a 2.5×117 cm BioGel P10 column (Bio-Rad,Hercules, Calif.) and eluted at 4° C. with 5% acetic acid.

[0178] Fractions containing 6.6 ml were obtained. Fractions were assayedby absorption at 280 nm and the elution pattern is shown in FIG. 1.

[0179] Aliquots (66 μl) of each fraction were dried by vacuumcentrifugation and resuspended in 6.6 μl of 0.01% acetic acid. Five μlsamples of this concentrate were tested for antimicrobial activityagainst E. coli ML-35, L. monocytogenes, strain EGD and C. albicans,strain 820, using radiodiffusion and gel overlay techniques as describedby Lehrer, R. I. et al. J Immunol Meth (1991) 137:167-173. Briefly, theunderlay agars used for all organisms had a final pH of 6.5 andcontained 9 mM sodium phosphate/1 mM sodium citrate buffer, 1% w/vagarose and 0.30 μg/ml tryptocase soy broth powder (BBL Cockeysville,Md.). The units of activity in the radial diffusion assay were measuredas described; 10 units correspond to a 1 mm diameter clear zone aroundthe sample well. Activities obtained for the various fractions are shownin FIG. 2. Activity was found in a large number of fractions.

[0180] The active fractions were further examined by acid-urea PAGE(AU-PAGE) and SDS PAGE. Results of these analyses showed that activeantimicrobial peptides of the appropriate molecular weight were presentand concentrated in fractions 76-78.

[0181] Fractions 76-78 from the Biogel P10 column were then pooled andchromatographed on a 1×25 cm Vydac 218 TP1010 column with a gradient(buffer A is 0.1% TFA; buffer B is 0.1% TFA in acetonitrile) theincrease in acetonitrile concentration was 1% per minute. The results,assessed in terms of absorbance at 280 nm and at 225 nm are shown inFIG. 3. The peaks corresponding to the three peptides illustrated hereinare labeled in the figure. The figure also contains an inset which showsthe results of an acid-urea PAGE gel stained with Comassie Blue thatcontains a starting mixture composed of the pooled fractions and theindividual PG species. These are labeled M, 1, 2 and 3 on the inset. Theresults clearly show the presence of three distinct proteins.

[0182] The isolated proteins were subjected to amino acid analysis usingthree independent methods, and to Edman degradation, chymotrypsindigestion, and fast atom bombardment mass spectrometric analysis. Thepeptides, named “protegrins”, are shown to,have the amino acid sequencesas follows: PG-1: RGGRLCYCRRRFCVCVGR PG-2: RGGRLCYCRRRFCICV PG-3:RGGGLCYCRRRFCVCVGR,

[0183] and are amidated at the C-terminus.

[0184] The amidation status of the isolated peptides was established bysynthesis of PG-3 both in the free carboxyl and carboxyamidated forms.These synthetic peptides were then compared to isolated PG-3 usingAU-PAGE and also using reverse-phase HPLC. In both cases, the nativeproduct comigrated with the synthetic amidated form.

[0185] The location of the disulfide linkages in the isolated protegrinswas also studied using PG-2 as a model. The determination was performedusing sequential enzyme digestion (chymotrypsin followed by thermolysin)with direct analysis using LC-ESI-MS on the fragments obtained. Theresults of these analyses showed that the two intramolecular disulfidebonds were C₆-C₁₅ and C₈-C₁₃. With the location of the disulfides inthese positions, the protegrin molecules are likely to exist asanti-parallel β sheets similar to the tachyplesins in overallconformation.

[0186] The antimicrobial proteins above are present in much lowerconcentrations in initial extracts than are the rabbit defensins incorresponding crude extracts where the defensins constitute more than15% of the total protein in rabbit granulocytes. Using the AU-PAGEanalytical method on the various stages of purification, the peptidesare only faintly visible in the crude extracts, whereas correspondingcrude extracts of rabbit granulocytes clearly show the presence of thedefensins. The peptides of the invention become clearly evident onlyafter the ultrafiltration step.

[0187] Because the protegrins whose structures are set forth above showsequence homology to the decapeptide region corresponding to residues1-10 of rabbit defensin NP-3a in the decapeptide region at positions4-13 of PG-3, the protegrins, and in particular PG-3, may share theproperty of defensin NP-3a in being capable of competitivelyantagonizing ACTH-mediated steroid synthesis by adrenocytes. Thisproperty, called “corticostasis”, may influence the effectiveness of theprotegrins as antiinfectious agents when employed in vivo.

EXAMPLE 2 Antimicrobial Activity

[0188] The radial diffusion assay in agarose gels described in Example 1was also used to test the activity of the purified protegrins. FIGS. 4a,4 b and 4 c show the results against three test organisms in unitsdescribed as above. The rabbit defensin (NP-1) and the human defensin(HNP-1) were used as controls.

[0189]FIG. 4a shows that PG-1 and PG-3 are more effective against E.coli ML-35P than HNP-1 and only slightly less effective than NP-1. PG-1and PH-3 were also effective against Listeria monocytogenes, strain EGDas shown in FIG. 4b. In FIG. 4c, PG-1 and PG-3 were also shown effectiveagainst Candida albicans. In general, these peptides are approximatelyas effective as rabbit defensin NP-1 on a weight basis and are moreeffective than HNP-1. In all cases, PG-2 was also effective against thethree organisms tested but was not as active as the other two peptides.

[0190] In addition to its activity in inhibiting the growth of theabove-mentioned organisms, the PG-1 of the invention has been showndirectly to inhibit the growth of Staphylococcus aureus (see Figure) andK. pneumoneae 270 (Figure). HNP-1 used as a control was less effectiveagainst S. aureus and almost entirely ineffective against K. pneumoneae.

[0191] The protegrins of the invention have also been tested againstvarious other organisms and show broad spectrum activity. In addition totheir effectiveness in inhibiting the growth of or infection bymicroorganisms associated with STDs as described in Example 9hereinbelow, the protegrins show strong activity against the followingmicroorganisms in addition to those tested hereinabove: Pseudomonasaeruginosa, Klebsiella pneumoniae, Salmonella typhimurium,Staphylococcus aureus, Histoplasma capsulatum, Myobacteriumavium-intracellulare, and Mycobacterium tuberculosis. The protegrinsshowed only fair activity against Vibrio vulnificus and were inactiveagainst Vibrio cholerae and Borrelia burgdorferi.

EXAMPLE 3 Retention of Activity Under Certain Conditions

[0192] The antimicrobial activity of the invention compounds was testedas set forth above, but under conditions of 100 μM NaCl and in thepresence of 90% fetal calf serum. FIGS. 5a and 5 b show that PG-1 andPG-3 retain their activity with respect to C. albicans and E. colirespectively, even in the presence of 100 mM NaCl. Neither NP-1 norHNP-1 have this property. FIG. 5c shows that although NP-1 and NHP-2lose their ability to inactivate C. albicans in 90% fetal calf serum,inactivation by PG-3 is retained.

[0193] Accordingly, the protegrins of the invention retain theirantimicrobial properties under useful physiological conditions,including isotonic and borate solutions appropriate for use in eye careproducts.

[0194] In addition, synthetic PG-1 was tested with respect to itsactivity against E. coli ML-35 (serum sensitive) in underlayered gelscontaining only 10 mM sodium phosphate buffer, pH 7.4 and a 1:100dilution of trypticase soy broth, both in the presence and absence of2.5% normal human serum, which is below the lytic concentration for thisstrain of E. coli. In the presence of serum, the minimal bacteriocidalconcentration was reduced from approximately 1.0 μg/ml to about 0.1μg/ml. This type of effect was not observed either for a linear fragmentof cathepsin G or for the defensin HNP-1.

[0195] Similarly, using C. albicans as a target organism, underlayerswere prepared with 10 mM sodium phosphate with and without 10% normalhuman serum. The minimal fungicidal concentration fell from about 1.3μg/ml in the absence of serum to 0.14 μg/ml in its presence. The serumitself at this concentration did not effect C. albicans.

[0196] Thus, not only is the action of the protegrins not inhibited bythe presence of serum, it is enhanced thereby. Similar results wereobtained using L. monocytogenes as the target organism.

[0197] The protegrins PG-1 and PG-3 were incubated for 4 hours at pH 2.0with 0.5 μg/ml pepsin and then neutralized. The residual antimicrobialactivity against C. albicans, E. coli and L. monocytogenes was assessedand found to be fully retained. Similar experiments show that thesecompounds are not degraded by human leukocyte elastase or by humanleukocyte cathepsin G even when exposed to high concentrations of theseenzymes and at a pH of 7.0-8.0 favorable for proteolytic activity. Inaddition, synthetic PG-3 amide and synthetic PG-3 acid were autoclavedand tested for antimicrobial activity against E. coli, L. monocytogenesand C. albicans; retaining full antimicrobial activity in all cases. Itis possible that the stability of these compounds to proteasedegradation and to autoclaving is enhanced by the presence of disulfidebonds.

EXAMPLE 4 Ability to Bind Endotoxin

[0198] The protegrins of the invention were tested for their ability tobind the lipid polysaccharide (LPS) of the gram-negative bacterium E.coli strain 0.55B5. The assay was the Limulus amebocyte lysate (LAL)test for endotoxins conducted in the presence and absence of the testcompounds. The test was conducted using the procedure described in SigmaTechnical Bulletin No. 210 as revised in December 1992 and published bySigma Chemical Company, St. Louis, Mo.

[0199] The LAL test is based on the ability of LPS to effect gelation inthe commercial reagent E-Toxate™ which is prepared from the lysate ofcirculating amebocytes of the Horseshoe Crab Limulus polyphemus. Asdescribed in the technical bulletin, when exposed to minute quantitiesof LPS, the lysate increases in opacity as well as viscosity and may geldepending on the concentration of endotoxin. The technical bulletin goeson to speculate that the mechanism appears analogous to the clotting ofmammalian blood and involves the steps of activation of a trypsin-likepreclotting enzymes by the LPS in the presence of calcium ion, followedby enzymic modifications of a “coagulogen” by proteolysis to produce aclottable protein. These steps are believed tied to the biologicallyactive or “pyrogenic” portion of the molecule. It has been shownpreviously that detoxified LPS (or endotoxin) gives a negative LAL test.

[0200] The test compounds were used at various concentrations from 0.25μg-10 μg in a final volume of 0.2 ml and the test mixtures contained LPSat a final concentration of 0.05 endotoxin unit/ml and E-Toxate™ at thesame concentration. The test compounds were incubated together with theLPS for 15 minutes before the E-Toxate™ was added to a final volumeafter E-Toxate™ addition of 0.2 ml. The tubes were then incubated for 30minutes at 37° C. and examined for the formation of a gel.

[0201] Both isolated native protegrins (nPGs) and synthetically preparedprotegrins (sPGs) were tested. The sPGs were prepared with a carboxylgroup at the C-terminus or with an amidated C-terminus. The nPGs areamidated at the C-terminus. Also tested were six different rabbitdefensins (NPs) and four native human defensins (HNPs). The results areshown in Table 1. TABLE 1 Peptide 10 μg 5 μg 2.5 μg 1.0 μg 0.5 μg 0.25μg nPG-1 no gel no gel no gel no gel + ++ nPG-2 no gel no gel no gel nogel + ++ nPG-3 no gel no gel trace ++ ++ ++ sPG-3 acid no gel no geltrace ++ ++ ++ sPG-3 amide no gel no gel no gel + ++ ++ NP-1 not not ++++ ++ ++ tested tested NP-2 trace + + ++ ++ ++ NP-3a no gel no gel nogel ++ ++ ++ NP-3b no gel no gel + ++ ++ ++ NP-4 not not + ++ ++ ++tested tested NP-5 no gel trace + + ++ ++ HNP-1 no gel + + ++ ++ ++HNP-2 trace trace trace + + ++ HNP-3 no gel + + ++ ++ ++ HNP-4 no geltrace trace ++ + ++

[0202] As seen from the results, all of the protegrins, both syntheticand native, and both in the amidated and nonamidated forms are able tobind sufficiently to LPS to prevent any substantial gel formation atconcentrations as low as 2.5 μg/0.2 ml. nPG-1 and nPG-2 are effective atsomewhat lower concentrations. The protegrins were substantially moreeffective than the NP or HNP test compounds; the most effective amongthese controls was NP-3a, a peptide whose primary sequence most closelyresembles that of the protegrins.

[0203] In a follow-up experiment, the concentration of LPS was variedfrom 0.05-0.25 endotoxin units (E.U.) and synthetic PG-3 amide was usedas the test compound. The results are shown in Table 2. TABLE 2Endotoxin Units 0.25 E.U. 0.10 E.U. 0.05 E.U. sPG-3 amide (2.5 Tg) nogel no gel no gel sPG-3 amide (1.0 Tg) no gel no gel no gel sPG-3 amide(0.5 Tg) ++ ++ no gel no added protein ++ ++ ++

[0204] These results show that since inhibition of gelation can beovercome by increasing the concentration of LPS, interaction with LPS isresponsible for the lack of gelation, rather than interfering with thegelation enzyme cascade.

EXAMPLE 5 Activity of Linearalized Forms

[0205] nPG-1 and nPG-3 were converted to linear form using a reducingagent to convert the disulfide linkages to sulfhydryl groups, which werethen stabilized by alkylating with iodoacetamide.

[0206] The ability of both cyclic and linearalized PG-1 and PG-3 toinhibit gelation in the standard LAL assay was assessed then asdescribed in Example 4 and the results are shown in Table 3. TABLE 3Peptide 5 μg 2.5 μg 1.0 μg 0.25 μg nPG-1 no gel no gel ++ ++ ++cam-nPG-1 no gel no gel ++ ++ ++ nPG-3 no gel no gel ++ ++ ++ cam-nPG-3no gel no gel ++ ++ ++

[0207] These results show that the linearalized and cyclic forms of theprotegrins are equally capable of inhibiting gelation and binding toendotoxin.

[0208] The antimicrobial activity of the linearalized forms was alsocompared with that of the native protegrins. Both linearalized andcyclic forms of the protegrins tested continue to show antimicrobialactivity, although the effectiveness of these peptides as antimicrobialsdepends on the nature of the target organism and on the test conditions.The antimicrobial activity of native PG-1 and its linearalized form(cam-PG-1) and PG-3 and its linearalized form (cam-PG-3) were testedaccording to the procedure set forth in Example 1 as described byLehrer, R. I. et al. J Immunol Meth (1991) 137:167-173. The results areset forth in FIGS. 6a-6 f.

[0209]FIGS. 6a and 6 b show the antimicrobial activity of these peptidesin the concentration range 20 μg/ml-125 μg/ml with respect to E. coliML-35P either in 10 mM phosphate-citrate buffer, pH 6.5 (FIG. 6a) or inthe presence of this buffer plus 100 mM NaCl (FIG. 6b). Both protegrinsshowed strong antimicrobial activity with respect to this organism; thelinear form was slightly more potent in the presence of buffer alonethan was the cyclic form; on the other hand, the cyclic form was morepotent than the linear form under isotonic conditions.

[0210]FIGS. 6c and 6 d show the antimicrobial effect with respect to L.monocytogenes. In FIG. 6c where the above-mentioned buffer alone wasused, both cyclic and linearalized forms of the protegrins showed strongantimicrobial activity and both were approximately equally effectiveover the concentration range tested (20 μg/ml-125 μg/ml).

[0211]FIG. 6d shows the effect with respect to L. monocytogenes in thepresence of this buffer plus 100 mM NaCl over the same concentrationrange. The cyclic form retained strong antimicrobial activity with aslightly greater concentration dependence. Linearalization appeared tolower the activity appreciably although high concentrations were stillable to show an antimicrobial effect.

[0212] The yeast C. albicans was tested with the results shown in FIGS.6e and 6 f. FIG. 6e shows that all forms of these protegrins wereantimicrobial in a dose-dependent manner over the above concentrationrange when tested in the presence of 10 mM phosphate buffer alone,although the linearalized peptides were very slightly less effective.FIG. 6f shows the results of the same assay run in the presence ofbuffer plus 100 mM NaCl. While the cyclized forms retained approximatelythe same level of antimicrobial effect, the activity of the linearalizedforms was greatly diminished so that at concentrations below 100 μg/mlof the protegrin, virtually no antimicrobial effect was seen. However,at higher concentrations of 130 μg/ml, a moderate antimicrobial effectwas observed.

[0213] Thus, depending on the target microorganism and the conditionsused, both the cyclized and linearalized forms of the protegrins haveantimicrobial activity.

EXAMPLE 6 Antimicrobial Activity Under Conditions Suitable for Treatmentof the Eye

[0214] Contact lens solutions are typically formulated with boratebuffered physiological saline and may or may not contain EDTA inaddition. Protegrins in the form of the synthetic PG-3 amide andsynthetic PG acid were tested generally in the assay described inExample 1 wherein all underlay gels contain 25 mM borate buffer, pH 7.4,1% (v/v) tryptocase soy broth (0.3 μg/ml TSB powder) and 1% agarose.Additions included either 100 mM NaCl, 1 mM EDTA or a combinationthereof. Other test compounds used as controls were the defensin NP-1and lysozyme. Dose response curves were determined.

[0215] Table 4 shows the estimated minimal bacteriocidal concentrationsin μg/ml of the various test compounds. TABLE 4 ESTIMATED MINIMALFUNGICIDAL CONCENTRATIONS (μg/ml) Peptide buffer +EDTA +NaCl +EDTA &NaCl sPG-3 amide 13.0 9.5 4.1 3.1 sPG-3 acid 15.0 9.5 4.6 3.7 NP-1 35.045.0 >200 >200 lysozyme 75.0 45.0 >200 >200

[0216] Although protegrins are somewhat less active in 25 mM boratebuffered saline than in 25 mM phosphate buffer, the antimicrobialactivity is enhanced by adding physiological saline and modestlyenhanced by 1 mM EDTA, as shown in the table.

[0217] A similar test was run with Candida albicans as the targetorganism with the results shown in Table 5, which also shows estimatesof minimal fungicidal concentrations. TABLE 5 ESTIMATED MINIMALFUNGICIDAL CONCENTRATIONS (μg/ml) 25 mM borate buffer + borate buffer +Peptide borate buffer 120 mM NaCl EDTA & NaCl nPG-3 32.0 9.0 8.0 sPG-3amide 19.0 7.7 7.0 sPG-3 acid 19.0 9.2 9.3 NP-1 23.0 60.0 65.0 HNP-125.0 >200 >200

[0218] Table 6 shows results of similar experiments conducted with L.monocytogenes as the target. TABLE 6 ESTIMATED MINIMAL BACTERICIDALCONCENTRATIONS (μg/ml) 25 mM borate buffer + borate buffer + Peptideborate buffer 120 mM NaCl EDTA & NaCl nPG-3 25.0 7.0 5.7 sPG-3 amide21.0 5.7 5.2 sPG-3 acid 30.0 7.0 7.0 NP-1 20.0 11.0 3.8 HNP-1 11.0 >200>200

[0219] The results shown indicate that these compounds are capable ofexerting their antimicrobial effects under conditions typicallyassociated with conditions suitable for eye care products.

EXAMPLE 7 Recovery of cDNA Clones and of a New Protegrin-Encoding cDNAcDNA Generation and PCR Amplification

[0220] Total RNA was extracted from the bone marrow cells of a young redDuroc pig with guanidinium thiocyanate. One μg of total RNA was used tosynthesize the first strand cDNA, with 20 pmol Oligo(dT) primer and 200U Moloney-murine leukemia virus (M-MLV) reverse transcriptase (ClontechLaboratory, Palo Alto, Calif.) in a total reaction volume of 20 μl. TwoPCR primers were prepared. The sense primer (5′-GTCGGAATTCATGGAGACCCAGAG(A or G) GCCAG-3′) corresponded to the 5′ regions of PG-2 and PR-39 cDNAand contained an EcoRI restriction site. The antisense primer(5′-GTCGTCTAGA (C or G) GTTTCACAAGAATTTATTT-3′) was complementary to 3′ends of PG-2 and PR-39 cDNA immediately preceding their poly A tails andcontained an XbaI restriction site. PCR was carried out in a 50 μlvolume using {fraction (1/10)} volume of the above pig cDNA as template,25 pmol primers and 2.5 units of AmpliTaq DNA polymerase (PerkinElmer-Cetus). The reaction was run for 30 cycles, with 1 mindenaturation (94° C.) and annealing (60° C.) steps and a 2 min extensionstep (72° C.) per cycle.

[0221] cDNA Cloning and Sequencing. The amplified cDNA was fractionatedby preparative agarose electrophoresis and stained with ethidiumbromide. The main fragment was cut out, digested with EcoR I and Xba Iendonucleases (New England Biolabs, Beverly, Mass.), subcloned into aM13mp18 bacteriophage vector, and transformed into E. coli XL1-Blue MRF′competent cells (Stratagene, La Jolla, Calif.). DNA sequencing wasperformed with a kit (U.S. Biochemical Corp., Cleveland, Ohio).Nucleotide and protein sequences were analyzed with PC-GENE(Intelligenetics, Palo Alto, Calif.).

[0222] Northern blots. Ten μg of total RNA was denatured in 50%formamide, separated by electrophoresis through 1% agarose gels in 0.62M formaldehyde, and blotted onto GeneScreen Plus membranes (DuPont,Boston, Mass.) by capillary transfer. The membrane was baked at 80° C.for 2 h, and hybridized with ³²P-labeled probe in rapid hybridizationbuffer (Amersham, Arlington Height, Ill.).

[0223] The results of sequencing the various clones encoding the variousprotegrins is summarized in FIG. 7. The cDNA sequences of protegrinsPG-1, PG-3 and PG-4 contain 691 bases as had previously been shown forPG-2 by Storici, P. et al. Biochem Biophys Res Comm (1993)196:1363-1368. The cDNAs show an upstream sequence encoding 110 aminoacids which appears identical for all protegrins. Additionaldifferences, which are quite slight in nature, are shown in FIG. 7.

[0224] The analysis showed the presence of the protegrin PG-4 having anamino acid sequence of Formula (1) wherein A₁₀ is a small amino acid andA₁₁ is a hydrophobic amino acid as distinguished from the previouslyknown protegrins where these residues are basic. The amino acid sequenceof PG-4 is therefore RGGRLCYCRGWICFCVGRG, wherein 1, 2, or 3 amino acidsat the N-terminus may be deleted.

[0225] Additional clones were obtained by amplifying reverse transcribedporcine bone cell RNA using an upstream primer that corresponds to the5′ end of PG-2 and another cathelin-associated peptide, PR39, (AgerbethB et al., Eur J Biochem (1991) 202:849-854; Storici, P et al., BiochemBiophys Res Com (1993) 186:1058-1065) and downstream primer that matchesthe region immediately preceding the poly A region. The resultingapproximately 0.7 kb PCR product was subcloned into M13mp18 andrecombinant plaques were chosen for purification and sequencing. In thismanner, the sequences for the precursors of PG-1, PG-3 and PG-4 wererecovered. All of these peptides are encoded by a nucleotide sequencewhich encodes a precursor containing additional amino acid sequenceupstream of A₁ of the compound of formula 1 (as shown for PG-4 in FIG.7).

EXAMPLE 8 Recovery of Genomic DNA Encoding PG-1, PG-3, and PG-5

[0226] High molecular genomic DNA was purified from pig white bloodcells with the QIAGEN blood DNA kit (QIAGEN, Chatsworth, Calif.). Toamplify protegrin (PG) genes, PCR was performed using genomic DNA as atemplate.

[0227] The sense primer (5′-GTCGGAATTCATGGAGACCCAGAG(A or G)GCCAG-3′)corresponded to the 5′ regions of PG cDNAs, of Example 7 and provided anEcoRI restriction site. The antisense primer (5′-GTCGTCTAGA(C orG)GTTTCACAAGAATTTATTT-3′) was complementary to 3′ ends of PG cDNAsimmediately preceding their poly(A) tails and provided an XbaIrestriction site. The reaction was carried out in a total volume of 50μl, which contained 200 ng of purified pig genomic DNA, 25 pmoles ofeach primer, 1 μl of 10 mM dNTP, 5 μl of 10×PCR buffer (200 mM Tris-HCl,100 mM(NH₄)₂, 20 mM MgSO₄, 1% Triton X-100, 0.1% BSA), and 2.5 units ofcloned Pfu DNA polymerase (Stratagene, La Jolla, Calif.). Thirty cycleswere performed, each with 1 min of denaturation at 94° C., 1 min ofprimer annealing at 55° C., 2 min of primer extension at 72° C., and afinal extension step at 72° C. for 10 min.

[0228] The amplified PCR product was digested with EcoRI and XbaI,excised from the agarose gel, purified, and ligated into pBluescript KS+vector (Stratagene, La Jolla, Calif.) that had been digested with EcoRIand XbaI and purified. Both strands of DNA were sequenced by the dideoxymethod using the Sequenase version 2.0 kit (United States Biochemical,Cleveland, Ohio), pBluescript universal primers and specific oligomerprimers based on PG genomic and cDNA sequences. Computer analysis of theDNA sequences was performed using the PC-Gene Program (Intelligenetics,Palo Alto, Calif.).

[0229] A PCR product of about 1.85 kb was confirmed as protegrin-relatedby hybridization with a protegrin-specific oligonucleotide probecomplementary to nt 403-429 of the protegrin cDNA sequences. The PCRproduct was then subcloned into pBluescript vector, and recombinantplasmids were subjected to DNA purification and sequencing. Genesequences for three different protegrins were identified PG-1, PG-3 andPG-5. The nucleotide sequences and deduced amino acid sequences areshown in FIG. 8.

[0230] Comparison of protegrin cDNAs and genes revealed that the codingregions of protegrin genes consisted of four exons, interrupted by threeintrons (FIGS. 8 and 9). The first exon contained the 5′ noncodingregion and codons for the first 66 amino acids of the protegrinprepropeptide, including a 29 residue signal peptide and the first 37cathelin residues. Exons II and III were relatively small, only 108 and72 bp respectively, and together contained the next 60 cathelinresidues. The final two cathelin residues were on Exon IV, and werefollowed by the protegrin sequences. The exon-intron splice sitesequences are shown in Table 7, and conform to the consensus rule: allintrons end on an AG doublet, preceded by a T/C rich stretch of 8-12bases, while all introns start with GT, followed predominantly by A/GA/G G sequence. TABLE 7 Exon-Intron Structure of the PG-1 Gene 5′ splice3′ splice Exon Size donor Intron Size acceptor 1 ? + AAGGCCgtgagtcg 1405 ttgaccagGACGAG 198 2 108 AACGGGgtgaggct 2 152 ccttccagCGGGTG 3 72AATGAGgtgagtgg 3 596 ggtcacagGTTCAA 4 313

[0231] The highly conserved cathelin region spans exons I-IV and Exon IVcontains the full sequence of the mature protegrin peptide followed byan amidation consensus sequence, a 3′ untranslated region, and theputative polyadenylation site. The three introns range in size from 152to 596 bp. If the protegrin genes are representative of othercathelin-like genes, the third intron of cathelin-associated peptideswill be found to separate all but the last two residues of the highlyconserved cathelin region from the variable antimicrobial peptidesencoded in Exon IV. Such a layout would favor recombination mechanismsinvolving association of diverse Exon IVs with the first three exonsspecifying cathelin containing prepro-regions.

[0232] The family of naturally occurring protegrins thus contains atleast 5 members. FIG. 10 shows a comparison of the amino acid sequencesof the five protegrins found so far in porcine leukocytes. There iscomplete homology in positions 1-3, 5-9, 13 and 15-16.

[0233] Homology search of protegrin genes against the EMBL/GenBankidentified no significantly homologous genes. More specifically, thegene structures and nucleotide sequences of protegrins were verydifferent from those of defensins, which contain three exons in myeloiddefensin genes, and two exons in enteric defensin genes. As expected,the search yielded the large family of cDNAs corresponding tocathelin-associated bovine, porcine and rabbit leukocyte peptides.

[0234] To assess protegrin-related genes further, we screened a porcinegenomic library of approximately 2.3×10⁵ clones in EMBL-3 SP6/T7 withthe ³²P-labeled protegrin cDNA, and identified 45 hybridizing clones.

[0235] A porcine liver genomic library in EMBL3 SP6/T7 phages waspurchased from Clontech (Palo Alto, Calif.). E. coli strain K803 wasused as a host, and DNA from phage plaques was transferred onto nylonmembranes (DuPont, Boston, Mass.). The filters were hybridized with³²P-labeled porcine 691 PG-3 cDNA. The filters were washed severaltimes, finally at 60° C. in 0.1×SSC and 0.1% SDS, and exposed to x-rayfilm with an intensifying screen at −70° C. Positive clones weresubjected to two additional rounds of plaque purification at lowdensity.

[0236] DNA purified from hybridizing clones was digested with variousrestriction endonucleases (New England Biolabs, Beverly, Mass.),fractionated on 0.8% agarose gels, and transferred onto GeneScreen Plusmembrane (DuPont, Boston, Mass.). The hybridization probes were labeledwith ³²P and included porcine PG-3 cDNA, and 5′-labeledprotegrin-specific oligonucleotide complementary to nt 403-429 of PG-1,2 and 3 cDNAs. For the cDNA probe, the hybridization and washingconditions were carried out as for the library screening. For theoligonucleotide probe, the membranes were washed at 42° C. in 0.1×SSC,0.1% SDS.

[0237] Southern blot analysis was carried out with purified DNA frompositive clones by hybridization with protegrin cDNA and a protegrinspecific oligonucleotide complementary to nt 403-429 of protegrin cDNAsequences. Although all of the clones hybridized with the complete cDNAprobe, only about half of them hybridized with the protegrin-specificprobe. A specific oligonucleotide probe for porcine prophenin, anothercathelin-associated porcine leukocyte-derived antimicrobial peptide,hybridized to several of the nonprotegrin clones. These results confirma) that the conserved proregion homologous to cathelin is present withinthe same gene as the mature antimicrobial peptides and is not added onby posttranscriptional events, and b) that the protegrins account forabout half of the cathelin-related genes in the pig.

[0238] A synthetic peptide corresponding to the amino acid sequence ofPG-5 was prepared and tested with respect to antimicrobial activityagainst E. coli, L. monocytogenes and C. albicans. The results werecompared to those obtained with a synthetically prepared PG-1. Theresults are shown in FIGS. 11a-11 c. As shown in these graphicalrepresentations of the results, PG-5 has comparable antimicrobialactivity to PG-1 against all three organisms tested.

EXAMPLE 9 Preparation of EnantioPG-1

[0239] Using standard solid phase techniques, a protegrin having theamino acid sequence of PG-1, but wherein every amino acid is in the Dform was prepared. This form of protegrin was tested against E. coli, L.monocytogenes, C. albicans and other microbes in the absence andpresence of protease and otherwise as described for the radiodiffusionassay in agarose gels set forth in Example 1. The results are shown inFIGS. 12a-12 g.

[0240]FIG. 12a shows that both native PG-1 and enantioPG-1 in theabsence of protease are equally effective in inhibiting the growth of E.coli. FIG. 12b shows that neither trypsin nor chymotrypsin inhibits theantibacterial effect of enantioPG-1. FIG. 12c shows that in the presenceof these proteolytic enzymes, the ability of native PG-1 to inhibit thegrowth of L. monocytogenes is adversely affected, although, as shown inFIG. 12d, in the absence of these proteases PG-1 is comparably active toan enantioPG-1.

EXAMPLE 10 Activity of the Protegrins Against STD Pathogens

[0241] Table 8 summarizes the activity of the protegrin PG-1 as comparedto the defensin HNP-1 against growth of STD pathogens. In these results,“active” means that the peptide was effective at less than 10 μg/ml;moderately active indicates that it was active at 10-25 μg/ml; andslightly active means activity at 25-50 μg/ml. If no effect was obtainedat 50-200 μg/ml the compound was considered inactive. TABLE 8 Activityagainst human STD pathogens Protegrin PG-1 Defensin HNP-1 HIV-1 ActiveSlightly active Chlamydia trachomatis Active Slightly active Treponemapallidum Active Inactive Neisseria gonorrhoeae Active InactiveTrichomonas vaginalis Moderately Inactive active Herpes simplex type 2Moderately Slightly active active Herpes simplex type 1 InactiveSlightly active Hemophilus ducreyi Not tested Not tested Human papillomavirus Not tested Not tested

[0242]Chlamydia trachomatis

[0243] Unlike other bacteria associated with STDs, Chlamydia requires anintracellular habitat for metabolic activity and binary fission. Thelife cycle is as follows: there is an extracellular form which is ametabolically inactive particle somewhat sporelike in its behavior,referred to as an elementary body (EB). The EB attaches to the host celland is ingested to form an internal vacuolar space often called an“inclusion”. The bacterium reorganizes to the delicate reticulate body(RB) which is noninfective but metabolically active and which over a48-72 hour period undergoes reformation to the EB state. The EBs arethen released from the cell. Rather than a peptidoglycan layer,Chlamydia contains multiple disulfide linkages in cysteine-rich proteinsfor protection in the EB stage.

[0244] The protegrins of the invention were tested for theirantimicrobial activity against Chlamydia using the “gold standard”chlamydial culture system for clinical specimens described by Clarke, L.M. in Clinical Microbiology Procedures Handbook II (1992), Isenberg, H.T. Ed. Am. Soc. Microbiol. Washington, D.C.; pp. 8.0.1 to 8.24.3.9.Briefly, McCoy cells (a mouse cell line) in cycloheximide EMEM with 10%fetal bovine serum (FBS) are used as hosts. Prior to chlamydialinoculation, the maintenance medium is aspirated without disruption ofthe cell layer and the cell layer is maintained on a cover slip in astandard vial. Each vial is then inoculated with 100-300 μL inoculum andcentrifuged at 3500×g for one hour at 20° C. The fluid is then aspiratedand 1 ml of EMEM is added. The vials are capped and incubated at 37° C.for 48 hours. After 48 hours the medium is again aspirated, coverslipsare rinsed twice with PBS and fixed with 300 μL EtOH for 10 minutes. TheEtOH is aspirated and the vials are allowed to dry; then one drop PBSplus 30 μL Syva Microtrak monoclonal antibody to the major outermembrane protein of Chlamydia is added for staining. After 37° C.incubation for 30 minutes, the cells are washed with distilled water andexamined for inclusions which are easily recognizable as bright,apple-green-staining cytoplasmic vacuoles. They represent the equivalentof a colony of free-living bacteria on standard bacterial culture media.

[0245] In the assays conducted below, C. trachomatis serovar L2(L2/434Bu) described by Kuo, C. C. et al. in Nongynococcal Urethritisand Related Infections (1977), Taylor-Robinson, D. et al. Ed. Am. Soc.Microbiol. Washington, D.C., pp. 322-326 was used. The seed is preparedfrom a sonicated culture in L929 mouse fibroblast cells, and partiallypurified by centrifugation. Since host protein is still present in theseed aliquots, each seed batch is titered at the time of preparationwith serial ten-fold dilutions to 2×10⁻⁹. The seed containing 9.2×10⁶IFU/ml is thawed quickly at 37° C. and diluted to 10⁻² withsucrose/phosphate salts/glycine to produce IFU of about 200 after roomtemperature preincubation and to dilute background eukaryotic protein.In the initial assays, the peptides to be tested were prepared as stocksolutions in 0.01% glacial acetic acid. 100 μL of the diluted chlamydialseed was aliquoted into 1.5 ml eppendorf tubes and 200 μL of theantibiotic peptide was added per tube. Aliquots of the peptide stock(and controls) were incubated with the seed at room temperature for onehour, two hours and four hours. About 10 minutes before the end of eachincubation period, maintenance media were aspirated from the McCoy vialsin preparation for standard inoculation and culture. Culture was thenperformed in the presence and absence of the peptides; in some cases,the peptides were added to final concentration in the culture media inaddition to the preculture incubation. The test was evaluatedmicroscopically.

[0246] The results using 50 μg of protegrin per addition were dramatic.In control cultures, where no peptides were added, 222-460 inclusionswere counted. In all protocols where protegrin was added either beforethe Chlamydia seed was added to the cells or both before and after, noinclusions were found. Similar results were obtained with 20 μgadditions of tachyplesin. The defensins NP-1 and HNP-1 had lesserprotective effects. In summary, the protegrins tested show antimicrobialagainst Chlamydia.

[0247] In the next series of experiments, various concentrations ofprotegrin (1 μg, 12.5 μg, 25 μg and 50 μg) were used in the two-hourpreincubation. Concentrations as low as 12.5 μg lowered the number ofinclusions to zero. Even at a concentration of 1 μg/ml, the number ofinclusions was lowered dramatically from about 110 to about 30.

[0248] In the next set of experiments, the effect of the presence ofserum was tested. The Chlamydia seed was preincubated for two hours withand without 10% FBS and also with or without protegrin at 25 μg.Protegrin was highly effective both with and without serum, whereashuman defensin HNP-2, used as a control, was reasonably effective in theabsence of serum but only marginally effective in its presence.

[0249] The experiments were repeated but adding 25 μg of protegrin oneafter the start of the chlamydial culture, i.e., after centrifugationand final medium mix and one hour into the beginning of the 48-hourculture period. Protegrin reduced the number of inclusions byapproximately 57% from untreated controls although HNP-2 was completelyineffective. Finally, the protegrin (at 25 μg) was added to thechlamydial seed and the mix then immediately cultured. In this case,without preincubation and without the one-hour post-infection gap,protegrin was minimally effective without or without serum.

[0250] The effect of serum is particularly important since for a topicalagent to be effective in combatting Chlamydia infection, it must act inthe presence of serum.

[0251] In addition, there are several mouse-based models for Chlamydiainfection which can be used to assess the efficacy of the protegrins.These include those described by Patton, D. L. et al. in ChlamydialInfections (1990) Bowie, W. R. et al. Eds. Cambridge University Press NYpp. 223-231; Swenson, C. E. et al. J. Infect. Dis. (1983) pp. 1101-1107,and Barron, A. L. et al. J. Infect. Dis. (1981) 143:63-66.

[0252]Neisseria gonorrhoeae

[0253] In more detail, the ability of the protegrins to inhibit N.gonorrhoeae was tested by a modification of the method of Miyasaki etal., Antimicrob Agent Chemother (1993) 37:2710-2715. Nonpiliatedtransparent variants of strains FA 19 and F 62 were propagated on GCBagar plates containing glucose and iron supplements overnight at 37° C.under 3.8% V/V CO2. These strains were chosen for their adaptability tothe assay.

[0254] The overnight growth is removed from the agar plate and suspendedin GCB broth containing supplements and sodium bicarbonate and grownwith shaking at 37° C. to mid log phase. The culture is diluted 1:100 inGCB broth to give about 10⁶ CFU/ml and serial dilutions were plated ontoGCB agar.

[0255] The peptides are dissolved in 0.01% v/v acetic acid to give a 1mg/ml stock solution and serially diluted. Ten μl of each dilution isadded to a sterile polystyrene tube containing 90 μl of dilutedbacteria-and the tubes are shaken at 37° C. for 45 minutes. The contentsare serially diluted 1:10 and plated on to GCB agar plates which areincubated in a CO₂ incubator. CFU are counted after 24 hours and the logbactericidal activity calculated.

[0256] Native PG-1, synthetic PG-1, synthetic PG-3 amide and syntheticPG-3 without amidation all gave over a 5 log reduction in CFU per ml inthis assay. Native PG-2 (containing 16 amino acids) gave a 2.6 foldreduction.

[0257] In addition enantioPG-1, the unidisulfide PG-1 (C₆-C₁₅), andunisulfide PG-1 (C₈-C₁₃) gave over a 5-fold log reduction in CFU/ml inthis assay.

[0258]Treponema pallidum

[0259] Bacteriocidal activity against this organism, which is theetiologic agent of syphilis, was also tested. Peptides were evaluated ata series of concentrations of 1.758 μg to 56.25 μg in 90 μl of unheatednormal rabbit serum. The serum served as a nutrient for the spirochetesto allow their survival during incubation as well as providing a sourceof complement. Ten μl of a suspension of T. pallidum containing about5×10⁷/μl organisms was added to each tube and the mixtures with theappropriate peptides were incubated at 34° C. under 95% N₂ and 5% CO₂.At time zero, just prior to incubation, 4 hours and 16 hours, 25randomly selected organisms were examined for the presence or absence ofmotility. The 50% immobilizing end point (IE₅₀) was calculated toindicate the concentration needed to immobilize 50% of the spirochetes.In the presence of PG-1, the IE₅₀ at 0 and 4 hours was 2.717 μg and<1.758 μg, respectively. Tachyplesin IE₅₀'s were 5.231 μg and 2.539 μgfor 0 and 4 hours. This was in contrast to HNP and NP preparations whichshowed little immobilizing ability.

[0260] Herpes Simplex Virus

[0261] Using viral stocks prepared in VERO cells, grown in minimalessential medium (MEM) with 2% fetal calf serum, the effect of variouspeptides on HSV 1 MacIntyre strain, a pool of ten clinical HSV 1isolates, HSV-2G, and a pool of ten clinical HSV 2 isolates, allsensitive to 3 μM acyclovir were tested. Two fibroblast cell lines,human W138 and equine CCL57, were used as targets and tests were done bydirect viral neutralization and delayed peptide addition.

[0262] In the direct neutralization format, the virus was preincubatedwith the peptides for 90 min before it was added to the tissue culturemonolayers. In the delayed peptide addition format, the virus was addedand allowed 50 min to adsorb to the target cells, then the monolayerswere washed and peptides were added for 90 min. Finally, the monolayerwas washed to remove the peptide and the cells were fed withpeptide-free MEM and cultured until the untreated infected monolayersexhibited 4+ cytopathic effect (CPE) (about 60 hours).

[0263] Antiviral activity was seen in both formats, but was morepronounced with the delayed peptide addition mode. In experimentsperformed with W138 and CCL57 cells in the direct neutralization format,PG-1 completely prevented HSV-2G from causing CPE at concentrations of50 μg/ml and 25 μg/ml, but these concentrations afforded no protectionagainst HSV-1, which produced 4+ CPE.

[0264] In the delayed peptide addition format, PG-1 completely preventedCPE by HSV-2G at 35 μg/ml and 50 μg/ml and it also fully protectedagainst the clinical HSV-2 pool at both concentrations.

[0265] Thus, PG-1 protected human and animal cells from infection bylaboratory and clinical strains of HSV-2, even when the peptides wereadded as late as 60 min after the virus had been introduced into thecell culture.

[0266]Trichomonas vaginallis

[0267]Trichomonas vaginallis strain C1 (ATCC 30001) was grown asdescribed by Gorrell, T. E. et al, Carlsberg Res Comm (1984) 49:259-268.In experiments performed in RPMI+1% heat-activated fetal calf serum,within a few minutes after exposure to 50 μg/ml PG-1, T. vaginallis(heretofore vigorously motile) became stationary. Soon thereafter, theorganisms became permeable to trypan blue, and, over the ensuing 15-30minutes, lysed. As expected, such organisms failed to grow whenintroduced into their customary growth medium (Diamond's medium).Organisms exposed to 25 μg/ml of PG-3 retained their motility.

[0268] Initial studies with two highly metronidazole-resistant clinicalisolates of T. vaginallis, strains MR and TV showed both weresusceptible to PG-1, including the C₈-C₁₃ and C₆-C₁₅ uni-disulfides andenantioPG-1 at concentrations of 100 and 50 μg/ml.

EXAMPLE 11 Antiretroviral Activity

[0269] Both synthetic and native PG-1 and native PG-2 were tested forantiviral activity against strains of HIV using the method described inMiles, S. A. et al., Blood (1991) 78:3200-3208. Briefly, the mononuclearcell fraction is recovered from normal donor leukopacs from the AmericanRed Cross using a Ficoll-hypaque density gradient. The mononuclear cellsare resuspended at 1×10⁶ cells per ml in RPMI 1640 medium with 20% fetalbovine serum, 1% penn/strep with fungizone and 0.5% PHA and incubated 24hours at 37° C. in 5% CO₂. The cells are centrifuged, washed and thenexpanded for 24 hours in growth medium.

[0270] Non-laboratory adapted, cloned HIV_(JR-CSF) and HIV_(JR-FL) wereelectroporated into the human peripheral blood mononuclear cellsprepared as described above. Titers were determined and in general,multiplicities of infection (MOI) of about 4,000 infectuous units percell are used (which corresponds to 25-40 picograms per ml HIV p24antigen in the supernatant).

[0271] In the assay, the HIV stocks prepared as above were diluted tothe correct MOI and the PBM are added to 24 well plates at aconcentration of 2×10⁶ per ml. One μl total volume is added to eachwell. The peptide to be tested is added in growth medium to achieve thefinal desired concentration. Then the appropriate number of MOI areadded. To assay viral growth, 200 μl of supernatant is removed on days 3and 7 and the concentration of p24 antigen is determined using acommercial assay (Coulter Immunology, Hialeah, Fla.). Controls includeduplicate wells containing cells alone, cells plus peptide at 5 μg/mlcells with virus but not peptide and cells with virus in the presence ofAZT at 10⁻⁵ M-10⁻⁸ M.

[0272] Using this assay, it was demonstrated that both natural andsynthetic PG-1 completely inhibit HIV infection at concentrationsbetween 1-5 μg/ml; IC₉₀ was <5 μg/ml. The time of addition of peptidewas then varied. Cells pretreated for 2 hours prior to addition ofvirus, at the time of addition of virus, or 2 hours after infectionshowed antiviral activity for the peptide. However, if PG-1 was added 24hours after infection, there was no antiviral activity.

[0273] Further, PG-2 shows similar activity but at a level approximately5-fold less. Alternative antibiotics such as human defensins and rabbitdefensins lacked potent activity in this assay. The results were similarfor both HIV_(JR-CSF) and HIV_(JR-FL) which are non-laboratory adaptedisolates (Koyanagi, Y. S. et al, Science (1987) 236:819-822).

[0274] The protegrins show similar activity with respect to otherretroviruses.

EXAMPLE 12 Preparation of Modified Protegrins: Kite and Bullet Forms

[0275] The kite and bullet forms of PG-1 wherein all X are alanine weresynthesized using conventional Fmoc chemistry. The crude syntheticpeptide was reduced by adding dithiothreitol (DTT) equal in weight tothe synthetic peptide which had been dissolved at 10 mg peptide/ml in asolution containing 6 molar guanidine HCl, 0.5 molar tris buffer, and 2mM EDTA, pH 8.05 and incubated for two hours at 52° C. under nitrogen.The mixture was passed through a 0.45 micron filter, acidified with{fraction (1/20)} (v/v) glacial acidic acid and subjected toconventional RP-HPLC purification with a C-18 column. HPLC-purified,reduced synthetic bullet and kite PG-1 were partially concentrated byvacuum centrifugation in a speed vac and allowed to fold for 24 hours atroom temperature in ambient air in 0.1 M Tris pH 7.7 at lowconcentration (0.1 mg peptide/ml) to minimize formation of interchaincystine disulfides. The mixture was then concentrated and acidified withHOAC to a final concentration of 5% and subjected to RP-HPLCpurification.

[0276] The purity of the final products bullet and kite PG-1 wasverified by AU-PAGE, analytical HPLC, and FAB-mass spec. AU-PAGE showeda single band for the final product in each case. The observed MH+ massvalues were 2093 in both cases.

EXAMPLE 13 Antimicrobial Activity of the Kite and Bullet Forms

[0277] The kite and bullet PG-1 compounds prepared in Example 12 weretested for antimicrobial activity using the radial diffusion assaydescribed in Example 1 as published by Lehrer, R. I. et al., J ImmunolMeth (1991) 137:167-173, except that the underlay agars contained 10 mmsodium phosphate buffer with a final pH of 7.4. As described in Example1, 0.3 mg/ml tripticase soy broth powder and 1% agarose were used aswell in the underlay agar. In some cases 100 mM NaCl or RPMI plus 2.5%normal human serum (NHS) was added to the agar.

[0278] In a first set of determinations, the bullet and kite forms ofPG-1 were tested for antimicrobial activity against L. monocytogenes, E.faecium (VR) or S. aureus under these three sets of conditions. FIG. 13shows the result.

[0279] As shown, the bullet and kite forms were roughly equallyeffective against these three bacteria using standard assay conditions.When 100 mM NaCl was added to the agar, however, the kite forms appearedslightly less active than the bullet forms which appear to have slightlyenhanced antimicrobial activity against all three stains except S.aureus under these conditions. Similarly, when RPMI plus 2.5% NHS wereadded, the bullet forms were again more effective than the kite forms.The activity of the kite form versus E. faecium was significantly lessunder these conditions.

[0280] As shown in FIG. 14, these forms of PG-1 were also tested againstE. coli, K. pneumoniae and P. aeruginosa. All three microorganisms wereinhibited by both kite and bullet forms under standard conditions. Thisantimicrobial activity was maintained also at 100 mM NaCl and RPMI plusNHS.

EXAMPLE 14 Synthesis of the Snake Form of PG-1

[0281] The snake form of PG-1 wherein all X are alanine was performedusing standard methods by Synpep Inc., Dublin, Calif. and the MH+ valuein FAB-mass spec was 2031.3 as expected. The snake form was purified tohomogeneity by RP-HPLC.

EXAMPLE 15 Antimicrobial Activity of Snake PG-1

[0282] Snake PG-1 was tested with respect to the same six organisms andusing the same conditions as set forth in Example 13 with respect to thebullet and kite forms of PG-1. The results are shown in FIGS. 15 and 16.In this case, the native two-cystine form of PG-1 (native) was used as acontrol. While the snake form shows somewhat superior activity withrespect to L. monocytogenes, E. faecium, and S. aureus under standardconditions, it is notably less effective than the native form in thepresence of either 100 mM NaCl or RPMI plus NHS. The same pattern isfollowed, as shown in FIG. 9 when the test organisms are E. coli, K.pneumoniae, and P. aeruginosa.

1 76 691 base pairs nucleic acid single linear CDS 1..450 1 ATG GAG ACCCAG AGA GCC AGC CTG TGC CTG GGG CGC TGG TCA CTG TGG 48 Met Glu Thr GlnArg Ala Ser Leu Cys Leu Gly Arg Trp Ser Leu Trp 1 5 10 15 CTT CTG CTGCTG GCA CTC GTG GTG CCC TCG GCC AGC GCC CAG GCC CTC 96 Leu Leu Leu LeuAla Leu Val Val Pro Ser Ala Ser Ala Gln Ala Leu 20 25 30 AGC TAC AGG GAGGCC GTG CTT CGT GCT GTG GAT CGC CTC AAC GAG CAG 144 Ser Tyr Arg Glu AlaVal Leu Arg Ala Val Asp Arg Leu Asn Glu Gln 35 40 45 TCC TCG GAA GCT AATCTC TAC CGC CTC CTG GAG CTG GAC CAG CCG CCC 192 Ser Ser Glu Ala Asn LeuTyr Arg Leu Leu Glu Leu Asp Gln Pro Pro 50 55 60 AAG GCC GAC GAG GAC CCGGGC ACC CCG AAA CCT GTG AGC TTC ACG GTG 240 Lys Ala Asp Glu Asp Pro GlyThr Pro Lys Pro Val Ser Phe Thr Val 65 70 75 80 AAG GAG ACT GTG TGT CCCAGG CCG ACC CGG CAG CCC CCG GAG CTG TGT 288 Lys Glu Thr Val Cys Pro ArgPro Thr Arg Gln Pro Pro Glu Leu Cys 85 90 95 GAC TTC AAG GAG AAC GGG CGGGTG AAA CAG TGT GTG GGG ACA GTC ACC 336 Asp Phe Lys Glu Asn Gly Arg ValLys Gln Cys Val Gly Thr Val Thr 100 105 110 CTG GAT CAG ATC AAG GAC CCGCTC GAC ATC ACC TGC AAT GAG GTT CAA 384 Leu Asp Gln Ile Lys Asp Pro LeuAsp Ile Thr Cys Asn Glu Val Gln 115 120 125 GGT GTC AGG GGA GGT CGC CTGTGC TAT TGT AGG CGT AGG TTC TGC GTC 432 Gly Val Arg Gly Gly Arg Leu CysTyr Cys Arg Arg Arg Phe Cys Val 130 135 140 TGT GTC GGA CGA GGA TGACGGTTGCGAC GGCAGGCTTT CCCTCCCCCA 480 Cys Val Gly Arg Gly * 145 150ATTTTCCCGG GGCCAGGTTT CCGTCCCCCA ATTTTTCCGC CTCCACCTTT CCGGCCCGCA 540CCATTCGGTC CACCAAGGTT CCCTGGTAGA CGGTGAAGGA TTTGCAGGCA ACTCACCCAG 600AAGGCCTTTC GGTACATTAA AATCCCAGCA AGGAGACCTA AGCATCTGCT TTGCCCAGGC 660CCGCATCTGT CAAATAAATT CTTGTGAAAC C 691 149 amino acids amino acid linearprotein 2 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg Trp Ser LeuTrp 1 5 10 15 Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala GlnAla Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu AsnGlu Gln 35 40 45 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp GlnPro Pro 50 55 60 Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser PheThr Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro ProGlu Leu Cys 85 90 95 Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val GlyThr Val Thr 100 105 110 Leu Asp Gln Ile Lys Asp Pro Leu Asp Ile Thr CysAsn Glu Val Gln 115 120 125 Gly Val Arg Gly Gly Arg Leu Cys Tyr Cys ArgArg Arg Phe Cys Val 130 135 140 Cys Val Gly Arg Gly 145 150 691 basepairs nucleic acid single linear CDS 1..444 3 ATG GAG ACC CAG AGA GCCAGC CTG TGC CTG GGG CGC TGG TCA CTG TGG 48 Met Glu Thr Gln Arg Ala SerLeu Cys Leu Gly Arg Trp Ser Leu Trp 155 160 165 CTT CTG CTG CTG GCA CTCGTG GTG CCC TCG GCC AGC GCC CAG GCC CTC 96 Leu Leu Leu Leu Ala Leu ValVal Pro Ser Ala Ser Ala Gln Ala Leu 170 175 180 AGC TAC AGG GAG GCC GTGCTT CGT GCT GTG GAT CGC CTC AAC GAG CAG 144 Ser Tyr Arg Glu Ala Val LeuArg Ala Val Asp Arg Leu Asn Glu Gln 185 190 195 TCC TCG GAA GCT AAT CTCTAC CGC CTC CTG GAG CTG GAC CAG CCG CCC 192 Ser Ser Glu Ala Asn Leu TyrArg Leu Leu Glu Leu Asp Gln Pro Pro 200 205 210 AAG GCC GAC GAG GAC CCGGGC ACC CCG AAA CCT GTG AGC TTC ACG GTG 240 Lys Ala Asp Glu Asp Pro GlyThr Pro Lys Pro Val Ser Phe Thr Val 215 220 225 230 AAG GAG ACT GTG TGTCCC AGG CCG ACC CGG CAG CCC CCG GAG CTG TGT 288 Lys Glu Thr Val Cys ProArg Pro Thr Arg Gln Pro Pro Glu Leu Cys 235 240 245 GAC TTC AAG GAG AACGGG CGG GTG AAA CAG TGT GTG GGG ACA GTC ACC 336 Asp Phe Lys Glu Asn GlyArg Val Lys Gln Cys Val Gly Thr Val Thr 250 255 260 CTG GAT CAG ATC AAGGAC CCG CTC GAC ATC ACC TGC AAT GAG GTT CAA 384 Leu Asp Gln Ile Lys AspPro Leu Asp Ile Thr Cys Asn Glu Val Gln 265 270 275 GGT GTC AGG GGA GGTCGC CTG TGC TAT TGT AGG CGT AGG TTC TGC ATC 432 Gly Val Arg Gly Gly ArgLeu Cys Tyr Cys Arg Arg Arg Phe Cys Ile 280 285 290 TGT GTC GGA TGAGGATGACGGT TGCGACGGCA GGCTTTCCCT CCCCCAATTT 484 Cys Val Gly * 295TCCCGGGGCC AGGTTTCCGT CCCCCAATTT TTCCGCCTCC ACCTTTCCGG CCCGCACCAT 544TCGGTCCACC AAGGTTCCCT GGTAGACGGA GAGGGATTTG CAGGCAACTC ACCCAGAAGG 604CCTTTCGGTA CATTAAAATC CCAGCAAGGA GACCTAAGCA TCTGCTTTGC CCAGGCCCGC 664ATCTGTCAAA TAAATTCTTG TGAAACC 691 147 amino acids amino acid linearprotein 4 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg Trp Ser LeuTrp 1 5 10 15 Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala GlnAla Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu AsnGlu Gln 35 40 45 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp GlnPro Pro 50 55 60 Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser PheThr Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro ProGlu Leu Cys 85 90 95 Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val GlyThr Val Thr 100 105 110 Leu Asp Gln Ile Lys Asp Pro Leu Asp Ile Thr CysAsn Glu Val Gln 115 120 125 Gly Val Arg Gly Gly Arg Leu Cys Tyr Cys ArgArg Arg Phe Cys Ile 130 135 140 Cys Val Gly 145 691 base pairs nucleicacid single linear CDS 1..450 5 ATG GAG ACC CAG AGA GCC AGC CTG TGC CTGGGG CGC TGG TCA CTG TGG 48 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu GlyArg Trp Ser Leu Trp 150 155 160 CTT CTG CTG CTG GCA CTC GTG GTG CCC TCGGCC AGC GCC CAG GCC CTC 96 Leu Leu Leu Leu Ala Leu Val Val Pro Ser AlaSer Ala Gln Ala Leu 165 170 175 180 AGC TAC AGG GAG GCC GTG CTT CGT GCTGTG GAT CGC CTC AAC GAG CAG 144 Ser Tyr Arg Glu Ala Val Leu Arg Ala ValAsp Arg Leu Asn Glu Gln 185 190 195 TCC TCG GAA GCT AAT CTC TAC CGC CTCCTG GAG CTG GAC CAG CCG CCC 192 Ser Ser Glu Ala Asn Leu Tyr Arg Leu LeuGlu Leu Asp Gln Pro Pro 200 205 210 AAG GCC GAC GAG GAC CCG GGC ACC CCGAAA CCT GTG AGC TTC ACG GTG 240 Lys Ala Asp Glu Asp Pro Gly Thr Pro LysPro Val Ser Phe Thr Val 215 220 225 AAG GAG ACT GTG TGT CCC AGG CCG ACCCGG CAG CCC CCG GAG CTG TGT 288 Lys Glu Thr Val Cys Pro Arg Pro Thr ArgGln Pro Pro Glu Leu Cys 230 235 240 GAC TTC AAG GAG AAC GGG CGG GTG AAACAG TGT GTG GGG ACA GTC ACC 336 Asp Phe Lys Glu Asn Gly Arg Val Lys GlnCys Val Gly Thr Val Thr 245 250 255 260 CTG GAT CAG ATC AAG GAC CCG CTCGAC ATC ACC TGC AAT GAG GTT CAA 384 Leu Asp Gln Ile Lys Asp Pro Leu AspIle Thr Cys Asn Glu Val Gln 265 270 275 GGT GTC AGG GGA GGT GGC CTG TGCTAT TGT AGG CGT AGG TTC TGC GTC 432 Gly Val Arg Gly Gly Gly Leu Cys TyrCys Arg Arg Arg Phe Cys Val 280 285 290 TGT GTC GGA CGA GGA TGACGGTTGCGAC GGCAGGCTTT CCCTCCCCCA 480 Cys Val Gly Arg Gly * 295ATTTTCCCGG GGCCAGGTTT CCGTCCCCCA ATTTTTCCGC CTCCACCTTT CCGGCCCGCA 540CCATTCGGTC CACCAAGGTT CCCTGGTAGA CGGTGAAGGA TTTGCAGGCA ACTCACCCAG 600AAGGCCTTTC GGTACATTAA AATCCCAGCA AGGAGACCTA AGCATCTGCT TTGCCCAGGC 660CCGCATCTGT CAAATAAATT CTTGTGAAAC C 691 149 amino acids amino acid linearprotein 6 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg Trp Ser LeuTrp 1 5 10 15 Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala GlnAla Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu AsnGlu Gln 35 40 45 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp GlnPro Pro 50 55 60 Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser PheThr Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro ProGlu Leu Cys 85 90 95 Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val GlyThr Val Thr 100 105 110 Leu Asp Gln Ile Lys Asp Pro Leu Asp Ile Thr CysAsn Glu Val Gln 115 120 125 Gly Val Arg Gly Gly Gly Leu Cys Tyr Cys ArgArg Arg Phe Cys Val 130 135 140 Cys Val Gly Arg Gly 145 150 691 basepairs nucleic acid single linear CDS 1..450 7 ATG GAG ACC CAG AGA GCCAGC CTG TGC CTG GGG CGC TGG TCA CTG TGG 48 Met Glu Thr Gln Arg Ala SerLeu Cys Leu Gly Arg Trp Ser Leu Trp 155 160 165 CTT CTG CTG CTG GCA CTCGTG GTG CCC TCG GCC AGC GCC CAG GCC CTC 96 Leu Leu Leu Leu Ala Leu ValVal Pro Ser Ala Ser Ala Gln Ala Leu 170 175 180 AGC TAC AGG GAG GCC GTGCTT CGT GCT GTG GAT CGC CTC AAC GAG CAG 144 Ser Tyr Arg Glu Ala Val LeuArg Ala Val Asp Arg Leu Asn Glu Gln 185 190 195 TCC TCG GAA GCT AAT CTCTAC CGC CTC CTG GAG CTG GAC CAG CCG CCC 192 Ser Ser Glu Ala Asn Leu TyrArg Leu Leu Glu Leu Asp Gln Pro Pro 200 205 210 AAG GCC GAC GAG GAC CCGGGC ACC CCG AAA CCT GTG AGC TTC ACG GTG 240 Lys Ala Asp Glu Asp Pro GlyThr Pro Lys Pro Val Ser Phe Thr Val 215 220 225 230 AAG GAG ACT GTG TGTCCC AGG CCG ACC CGG CAG CCC CCG GAG CTG TGT 288 Lys Glu Thr Val Cys ProArg Pro Thr Arg Gln Pro Pro Glu Leu Cys 235 240 245 GAC TTC AAG GAG AACGGG CGG GTG AAA CAG TGT GTG GGG ACA GTC ACC 336 Asp Phe Lys Glu Asn GlyArg Val Lys Gln Cys Val Gly Thr Val Thr 250 255 260 CTG GAT CAG ATC AAGGAC CCG CTC GAC ATC ACC TGC AAT GAG GTT CAA 384 Leu Asp Gln Ile Lys AspPro Leu Asp Ile Thr Cys Asn Glu Val Gln 265 270 275 GGT GTC AGG GGA GGTCGC CTG TGC TAT TGT AGG GGT TGG ATC TGC TTC 432 Gly Val Arg Gly Gly ArgLeu Cys Tyr Cys Arg Gly Trp Ile Cys Phe 280 285 290 TGT GTC GGA CGA GGATGA CGGTTGCGAC GGCAGGCTTT CCCTCCCCCA 480 Cys Val Gly Arg Gly * 295 300ATTTTCCCGG GGCCAGGTTT CCGTCCCCCA ATTTTTCCGC CTCCACCTTT CCGGCCCGCA 540CCATTCGGTC CACCAAGGTT CCCTGGTAGA CGGTGAAGGA TTTGCAGGCA ACTCACCCAG 600AAGGCCTTTC GGCACATTAA AATCCCAGCA AGGAGACCTA AGCATCTGCT TTGCCCAGGC 660CCGCATCTGT CAAATAAATT CTTGTGAAAC C 691 149 amino acids amino acid linearprotein 8 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg Trp Ser LeuTrp 1 5 10 15 Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala GlnAla Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu AsnGlu Gln 35 40 45 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp GlnPro Pro 50 55 60 Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser PheThr Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro ProGlu Leu Cys 85 90 95 Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val GlyThr Val Thr 100 105 110 Leu Asp Gln Ile Lys Asp Pro Leu Asp Ile Thr CysAsn Glu Val Gln 115 120 125 Gly Val Arg Gly Gly Arg Leu Cys Tyr Cys ArgGly Trp Ile Cys Phe 130 135 140 Cys Val Gly Arg Gly 145 150 1843 basepairs nucleic acid single linear DNA (genomic) CDS join(1..198,603..710, 863..934, 1531..1602) 9 ATG GAG ACC CAG AGA GCC AGC CTG TGCCTG GGG CGC TGG TCA CTG TGG 48 Met Glu Thr Gln Arg Ala Ser Leu Cys LeuGly Arg Trp Ser Leu Trp 1 5 10 15 CTT CTG CTG CTG GCA CTC GTG GTG CCCTCG GCC AGC GCC CAG GCC CTC 96 Leu Leu Leu Leu Ala Leu Val Val Pro SerAla Ser Ala Gln Ala Leu 20 25 30 AGC TAC AGG GAG GCC GTG CTT CGT GCT GTGGAT CGC CTC AAC GAG CAG 144 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val AspArg Leu Asn Glu Gln 35 40 45 TCC TCG GAA GCT AAT CTC TAC CGC CTC CTG GAGCTG GAC CAG CCG CCC 192 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu LeuAsp Gln Pro Pro 50 55 60 AAG GCC GTGAGTCGGG CAGGGGCTCA GGAGGGGCTGGGGGGCGGGG GCTGTCCCCC 248 Lys Ala 65 ACCCGCCCCG GGGCTCCCTG TCCCTCCCCCTGCTCAGGCT GTCCCTCCTG CCAGGAAGGC 308 ACTTGTCCCT CTAAGGGGGA CCCCCTCTGCCAGGAAACCT TCCCAGAGCT GGGTGCCCTG 368 CCCGCGTGAG AGCTTCCCGC CTTAGCCTCTGGGCTGTGGG CTCAGGGCCC TGCACAGCCT 428 GTGAGGCAGG AGCGGGCTCT GTCCCCTCCCCTGTGCACCC AGCACCAAGC CCAGGGCCAG 488 GCTCCCAGCA GGGGCTGCAG AGGCTGCTGTCTAGGTGGGG GCGGGGAGGG GGTGACAGAT 548 CCGAGGGGGA AGCCTGAGCC CGAGTCCCATCTCCCCACTT TGATCCTTGA CCAG GAC 605 Asp GAG GAC CCG GGC ACC CCG AAA CCTGTG AGC TTC ACG GTG AAG GAG ACT 653 Glu Asp Pro Gly Thr Pro Lys Pro ValSer Phe Thr Val Lys Glu Thr 70 75 80 GTG TGT CCC AGG CCG ACC CGG CAG CCCCCG GAG CTG TGT GAC TTC AAG 701 Val Cys Pro Arg Pro Thr Arg Gln Pro ProGlu Leu Cys Asp Phe Lys 85 90 95 GAG AAC GGG GTGAGGCTGG GGGCTGGGGGCGCTGGCGGA TGCTTCCCAA 750 Glu Asn Gly 100 GGAGCTGAAC AGGAGAGCCTGCTGGGGAAG ATGTCCAGGC CCTGGGGTGA GGCTGGGAGC 810 TCATGGATGG AGGAGGGGGGGTCCCAGTTT GACCTTGAGT CTCCCCTTCC AG CGG 865 Arg GTG AAA CAG TGT GTG GGGACA GTC ACC CTG GAT CAG ATC AAG GAC CCG 913 Val Lys Gln Cys Val Gly ThrVal Thr Leu Asp Gln Ile Lys Asp Pro 105 110 115 CTC GAC ATC ACC TGC AATGAG GTGAGTGGCC CCTTATTGGT GTCAAGTTGC 964 Leu Asp Ile Thr Cys Asn Glu 120125 TAATGGGTTG GTGTGGGGAA CTCCTTGGGA GTGTTACCCG CTGCCCCATC CAGGGCGTGG1024 AAAGGCCCTC CTACCCCGGC CCTTCCCTCA CCTCGGCCCC AGGGCTCCAG GTCTGGCTCT1084 GTCATCCTTA GGGCCGCGGT TCCCTCAATG GGGTCCCCCC CTCGTATTTG TCAGAAAGGC1144 ACATTTCAGG CCCCACCCCG ACCCTCTGAA TCACACTCTT GGGTGGAGCC CAGCCTTGTC1204 TCTTCTCCCA AGATCCCAGC GGGTTCTTCC TGTGCTGTCG GCTGAGAGGC AGTGACCGGA1264 CTAATGGACT TGCAGGCCCT GCTCCTGGCC AGCTTTGCGG GGCTGGGTTT GGGACCCTGG1324 CAAGGCCCCA GCCATCTCTG GGCCTGAGTC CACTTATGTG TCTGTGGGGG ATTCCACCAC1384 GTGCTCCAAA GGTCACAGCC AGAGGTGGAC CAGGGCCCCA AGCCTCTTAC TGTTTCCCCA1444 TTCAGGGATT TTTCTAGTCT GGAGGGAGGG TTCTTGTCTT GACCCTTGGC CAGACCCCAC1504 CCGAAACCTG TTTCTCTTGG TCACAG GTT CAA GGT GTC AGG GGA GGT CGC CTG1557 Val Gln Gly Val Arg Gly Gly Arg Leu 130 135 TGC TAT TGT AGG CGT AGGTTC TGC GTC TGT GTC GGA CGA GGA TGA 1602 Cys Tyr Cys Arg Arg Arg Phe CysVal Cys Val Gly Arg Gly * 140 145 150 CGGTTGCGAC GGCAGGCTTT CCCTCCCCCAATTTTCCCGG GGCCAGGTTT CCGTCCCCCA 1662 ATTTTTCCGC CTCCACCTTT CCGGCCCGCACCATTCGGTC CACCAAGGTT CCCTGGTAGA 1722 CGGTGAAGGA TTTGCAGGCA ACTCACCCAGAAGGCCTTTC GGTACATTAA AATCCCAGCA 1782 AGGAGACCTA AGCATCTGCT TTGCCCAGGCCCGCATCTGT CAAATAAATT CTTGTGAAAC 1842 C 1843 149 amino acids amino acidlinear protein 10 Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg TrpSer Leu Trp 1 5 10 15 Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala SerAla Gln Ala Leu 20 25 30 Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp ArgLeu Asn Glu Gln 35 40 45 Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu LeuAsp Gln Pro Pro 50 55 60 Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro ValSer Phe Thr Val 65 70 75 80 Lys Glu Thr Val Cys Pro Arg Pro Thr Arg GlnPro Pro Glu Leu Cys 85 90 95 Asp Phe Lys Glu Asn Gly Arg Val Lys Gln CysVal Gly Thr Val Thr 100 105 110 Leu Asp Gln Ile Lys Asp Pro Leu Asp IleThr Cys Asn Glu Val Gln 115 120 125 Gly Val Arg Gly Gly Arg Leu Cys TyrCys Arg Arg Arg Phe Cys Val 130 135 140 Cys Val Gly Arg Gly 145 150 18amino acids amino acid single linear Disulfide-bond 4..13 Disulfide-bond6..11 11 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val1 5 10 15 Gly Arg 16 amino acids amino acid single linear 12 Arg Gly GlyArg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Ile Cys Val 1 5 10 15 18 aminoacids amino acid single linear 13 Arg Gly Gly Gly Leu Cys Tyr Cys ArgArg Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acidsingle linear 14 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Gly Trp Ile Cys PheCys Val 1 5 10 15 Gly Arg 18 amino acids amino acid single linear 15 ArgPhe Phe Arg Leu Cys Tyr Cys Arg Pro Arg Phe Cys Val Cys Val 1 5 10 15Gly Arg 18 amino acids amino acid single linear 16 Arg Gly Gly Arg LeuCys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 16 aminoacids amino acid single linear 17 Arg Gly Gly Arg Leu Cys Tyr Cys ArgArg Arg Phe Cys Ile Cys Val 1 5 10 15 18 amino acids amino acid singlelinear 18 Arg Gly Gly Gly Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val CysVal 1 5 10 15 Gly Arg 18 amino acids amino acid single linear 19 Arg GlyGly Arg Leu Cys Tyr Cys Arg Gly Trp Ile Cys Phe Cys Val 1 5 10 15 GlyArg 18 amino acids amino acid single linear 20 Arg Gly Gly Arg Leu CysTyr Cys Arg Pro Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 16 amino acidsamino acid single linear 21 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg ArgPhe Cys Val Cys Val 1 5 10 15 16 amino acids amino acid single linear 22Lys Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 1015 16 amino acids amino acid single linear Modified-site 4 /product=“homoarginine(Har)” 23 Arg Gly Gly Xaa Leu Cys Tyr Cys Arg Arg Arg PheCys Val Cys Val 1 5 10 15 18 amino acids amino acid single linearModified-site group(4, 9) /product= “homoarginine(Har)” 24 Arg Gly GlyXaa Leu Cys Tyr Cys Xaa Arg Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Modified-site 10 /product=“homoarginine(Har)” 25 Arg Gly Gly Arg Val Cys Tyr Cys Arg Xaa Arg PheCys Val Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear 26 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Lys Lys Trp Cys Val CysVal 1 5 10 15 Gly Arg 18 amino acids amino acid single linearModified-site 10 /product= “homoarginine(Har)” 27 Arg Gly Gly Arg LeuCys Tyr Cys Arg Xaa Arg Tyr Cys Val Cys Val 1 5 10 15 Gly Arg 18 aminoacids amino acid single linear 28 Arg Gly Ser Gly Leu Cys Tyr Cys ArgArg Lys Trp Cys Val Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acidsingle linear 29 Arg Ala Thr Arg Ile Cys Phe Cys Arg Arg Arg Phe Cys ValCys Val 1 5 10 15 Gly Arg 18 amino acids amino acid single linearModified-site 10 /product= “homoarginine(Har)” 30 Arg Gly Gly Lys ValCys Tyr Cys Arg Xaa Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 18 aminoacids amino acid single linear Modified-site 9 /note= “D-form of aminoacid” Modified-site 18 /note= “D form of amino acid” 31 Arg Ala Thr ArgIle Cys Phe Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Modified-site 10 /product=“homoarginine(Har)” /note= “D form of amino acid” 32 Arg Gly Gly Lys ValCys Tyr Cys Arg Xaa Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 18 aminoacids amino acid single linear Region 1..18 /note= “All D-form aminoacids” 33 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val CysVal 1 5 10 15 Gly Arg 16 amino acids amino acid single linear Region1..16 /note= “All D-form amino acids” 34 Arg Gly Gly Arg Leu Cys Tyr CysArg Arg Arg Phe Cys Ile Cys Val 1 5 10 15 18 amino acids amino acidsingle linear Region 1..18 /note= “All D-form amino acids” 35 Arg GlyGly Gly Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 10 15 GlyArg 18 amino acids amino acid single linear Region 1..18 /note= “AllD-form amino acids” 36 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Gly Trp IleCys Phe Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear 37 Arg Gly Gly Arg Leu Val Tyr Cys Arg Arg Arg Phe Cys Val CysVal 1 5 10 15 Gly Arg 16 amino acids amino acid single linear 38 Arg GlyGly Arg Leu Gly Tyr Cys Arg Arg Arg Phe Cys Ile Cys Val 1 5 10 15 18amino acids amino acid single linear 39 Arg Gly Gly Gly Leu Cys Tyr GlyArg Arg Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 16 amino acids aminoacid single linear 40 Arg Gly Gly Arg Leu Gly Tyr Gly Arg Arg Arg PheGly Val Cys Val 1 5 10 15 16 amino acids amino acid single linear 41 LysGly Gly Arg Leu Val Tyr Val Arg Arg Arg Phe Ile Val Cys Val 1 5 10 15 16amino acids amino acid single linear Modified-site 4 /product=“homoarginine(Har)” 42 Arg Gly Gly Xaa Leu Cys Tyr Cys Arg Arg Arg PheCys Val Gly Val 1 5 10 15 18 amino acids amino acid single linearModified-site group(4, 9) /product= “homoarginine(Har)” 43 Arg Gly GlyXaa Leu Cys Tyr Cys Xaa Arg Arg Phe Cys Val Leu Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Modified-site 10 /product=“homoarginine(Har)” 44 Arg Gly Gly Arg Val Cys Tyr Val Arg Xaa Arg PheLeu Val Gly Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear 45 Arg Gly Gly Arg Leu Cys Tyr Ser Arg Lys Lys Trp Cys Val SerVal 1 5 10 15 Gly Arg 18 amino acids amino acid single linearModified-site 10 /product= “homoarginine(Har)” 46 Arg Gly Gly Arg LeuCys Tyr Cys Arg Xaa Arg Tyr Ser Val Val Val 1 5 10 15 Gly Arg 18 aminoacids amino acid single linear 47 Arg Gly Ser Gly Leu Ser Tyr Cys ArgArg Lys Trp Gly Val Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acidsingle linear 48 Arg Ala Thr Arg Ile Ser Phe Ser Arg Arg Arg Phe Ser ValSer Val 1 5 10 15 Gly Arg 18 amino acids amino acid single linearModified-site 10 /product= “homoarginine(Har)” 49 Arg Gly Gly Lys ValCys Tyr Gly Arg Xaa Arg Phe Ser Val Cys Val 1 5 10 15 Gly Arg 18 aminoacids amino acid single linear Modified-site group(9, 18) /note= “D formof amino acids” 50 Arg Ala Thr Arg Ile Val Phe Cys Arg Arg Arg Phe GlyVal Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acid single linearModified-site 10 /product= “homoarginine(Har)” /note= “D form of aminoacid” 51 Arg Gly Gly Lys Val Cys Tyr Leu Arg Xaa Arg Phe Leu Val Cys Val1 5 10 15 Gly Arg 18 amino acids amino acid single linear 52 Arg Gly GlyArg Ile Cys Phe Leu Arg Pro Arg Ile Gly Val Cys Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Disulfide-bond 8..13 Modified-sitegroup(6, 15) /note= “X is a hydrophobic, a small, or a large polar aminoacid” 53 Arg Gly Gly Arg Leu Xaa Tyr Cys Arg Arg Arg Phe Cys Val Xaa Val1 5 10 15 Gly Arg 18 amino acids amino acid single linear Disulfide-bond8..13 Modified-site group(6, 15) /note= “X is a hydrophobic, a small, ora large polar amino acid” 54 Arg Gly Gly Arg Leu Xaa Tyr Cys Arg Arg ArgPhe Cys Ile Xaa Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear Disulfide-bond 8..13 Modified-site group(6, 15) /note= “X is ahydrophobic, a small, or a large polar amino acid” 55 Arg Gly Gly GlyLeu Xaa Tyr Cys Arg Arg Arg Phe Cys Val Xaa Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Disulfide-bond 8..13 Modified-sitegroup(6, 15) /note= “X is a hydrophobic, a small, or a large polar aminoacid” 56 Arg Gly Gly Arg Leu Xaa Tyr Cys Arg Trp Gly Ile Cys Phe Xaa Val1 5 10 15 Gly Arg 18 amino acids amino acid single linear Disulfide-bond8..13 Modified-site group(6, 15) /note= “X is a hydrophobic, a small, ora large polar amino acid” 57 Arg Gly Gly Arg Leu Xaa Tyr Cys Arg Pro ArgPhe Cys Val Xaa Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear Disulfide-bond 6..15 Modified-site group(8, 13) /note= “X is ahydrophobic, a small, or a large polar amino acid” 58 Arg Gly Gly ArgLeu Cys Tyr Xaa Arg Arg Arg Phe Xaa Val Cys Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Disulfide-bond 6..15 Modified-sitegroup(8, 13) /note= “X is a hydrophobic, a small, or a large polar aminoacid” 59 Arg Gly Gly Arg Leu Cys Tyr Xaa Arg Arg Arg Phe Xaa Ile Cys Val1 5 10 15 Gly Arg 18 amino acids amino acid single linear Disulfide-bond6..15 Modified-site group(8, 13) /note= “X is a hydrophobic, a small, ora large polar amino acid” 60 Arg Gly Gly Gly Leu Cys Tyr Xaa Arg Arg ArgPhe Xaa Val Cys Val 1 5 10 15 Gly Arg 18 amino acids amino acid singlelinear Disulfide-bond 6..15 Modified-site group(8, 13) /note= “X is ahydrophobic, a small, or a large polar amino acid” 61 Arg Gly Gly ArgLeu Cys Tyr Xaa Arg Trp Gly Ile Xaa Phe Cys Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Disulfide-bond 6..15 Modified-sitegroup(8, 13) /note= “X is a hydrophobic, a small, or a large polar aminoacid” 62 Arg Gly Gly Arg Leu Cys Tyr Xaa Arg Pro Arg Phe Xaa Val Cys Val1 5 10 15 Gly Arg 18 amino acids amino acid single linear Modified-sitegroup(6, 8, 13, 15) /note= “X is a hydrophobic, a small, or a largepolar amino acid” 63 Arg Gly Gly Arg Leu Xaa Tyr Xaa Arg Arg Arg Phe XaaVal Xaa Val 1 5 10 15 Gly Arg 16 amino acids amino acid single linearModified-site group(6, 8, 13, 15) /note= “X is a hydrophobic, a small,or a large polar amino acid” 64 Arg Gly Gly Arg Leu Xaa Tyr Xaa Arg ArgArg Phe Xaa Ile Xaa Val 1 5 10 15 18 amino acids amino acid singlelinear Modified-site group(6, 8, 13, 15) /note= “X is a hydrophobic, asmall, or a large polar amino acid” 65 Arg Gly Gly Gly Leu Xaa Tyr XaaArg Arg Arg Phe Xaa Val Xaa Val 1 5 10 15 Gly Arg 18 amino acids aminoacid single linear Modified-site group(5, 7, 13, 15) /note= “X is ahydrophobic, a small, or a large polar amino acid” 66 Arg Gly Gly ArgXaa Leu Xaa Tyr Arg Gly Trp Ile Xaa Phe Xaa Val 1 5 10 15 Gly Arg 18amino acids amino acid single linear Modified-site group(6, 8, 13, 15)/note= “X is a hydrophobic, a small, or a large polar amino acid” 67 ArgGly Gly Arg Leu Xaa Tyr Xaa Arg Arg Arg Phe Xaa Val Xaa Val 1 5 10 15Gly Arg 30 base pairs nucleic acid single linear 68 GTCGGAATTCATGGAGACCC AGAGRGCCAG 30 30 base pairs nucleic acid single linear 69GTCGTCTAGA SGTTTCACAA GAATTTATTT 30 19 amino acids amino acid singlelinear 70 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Gly Trp Ile Cys Phe CysVal 1 5 10 15 Gly Arg Gly 14 base pairs nucleic acid single linear 71AAGGCCGTGA GTCG 14 14 base pairs nucleic acid single linear 72AACGGGGTGA GGCT 14 14 base pairs nucleic acid single linear 73AATGAGGTGA GTGG 14 14 base pairs nucleic acid single linear 74TTGACCAGGA CGAG 14 14 base pairs nucleic acid single linear 75CCTTCCAGCG GGTG 14 14 base pairs nucleic acid single linear 76GGTCACAGGT TCAA 14

What is claimed is:
 1. A purified and isolated or recombinantly producedcompound having the formula:A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁₀-A₁₁-A₁₂-C₁₃-A₁₄-C₁₅-A₁₆-A₁₇-A₁₈   (1) ora pharmaceutically acceptable salt or an N-terminal acylated orC-terminal amidated or esterified form thereof, which is either in alinear form or in a cystine-bridged form, wherein: each of A₁ and A₉ isindependently a basic amino acid; each of A₂ and A₃ is independently asmall amino acid; each of A₅, A₇, A₁₂, A₁₄ and A₁₆ is independently ahydrophobic amino acid; A₄ is a basic or a small amino acid; A₁₀ is abasic or a small amino acid or is proline; A₁₁ is a basic or ahydrophobic amino acid; A₁₇ is not present or, if present, is a smallamino acid; A₁₈ is not present or, if present, is a basic amino acid;and each of C₆, C₈, C₁₃ and C₁₅ is independently selected from the groupconsisting of cysteine, a hydrophobic amino acid, a large polar aminoacid and a small amino acid.
 2. The compound of claim 1 which has one ormore characteristics selected from the group consisting of: theC-terminal carboxyl is of the formula selected from the group consistingof COOH or salts thereof; COOR, CONH₂, CONHR and CONR₂ wherein each R isindependently a hydrocarbyl (1-6C); the amino group at the N-terminus isof the formula NH₂ or NHCOR wherein R is a hydrocarbyl (1-6C); each ofA₁ and A₉ is independently selected from the group consisting of R, Kand Har; each of A₂ and A₃ is independently selected from the groupconsisting of G, A, S and T; A₄ is R or G; each of A₅, A₁₄, and A₁₆ isindependently selected from the group consisting of I, V, NLe, L and F;each of A₇ and A₁₂ is independently selected from the group consistingof I, V, L, W, Y and F; A₁₀ is R, G or P; and A₁₁ is R or W.
 3. Thecompound of claim 1 which has antimicrobial or antiviral activityagainst pathogens associated with sexually transmitted disease.
 4. Thecompound of claim 1 which has antimicrobial or antiviral activityagainst Escherichia coli, Listeria monocytogenes, Candida albicans,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium,Staphylococcus aureus, Histoplasma capsulatum, Myobacteriumavium-intracellulare, Mycobacterium tuberculosis, Vibrio vulnificus,Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae,Trichomonas vaginalis, Herpes simplex virus type 1, Herpes simplex virustype 2, human immunodeficiency virus, Hemophilus ducreyi, or humanpapilloma virus.
 5. The compound of claim 1 which is selected from thegroup consisting of PG-1: RGGRLCYCRRRFCVCVGR; (SEQ ID NO:16) PG-2:RGGRLCYCRRRFCICV; (SEQ ID NO:17) PG-3: RGGGLCYCRRRFCVCVGR; (SEQ IDNO:18) PG-4: RGGRLCYCRGWICFCVGR; (SEQ ID NO:19) PG-5:RGGRLCYCRPRFCVCVGR; (SEQ ID NO:20)

and the amidated forms thereof either in linear or cystine-bridged form.6. A pharmaceutical composition comprising a compound according to claim1 and a pharmaceutically acceptable excipient.
 7. A method of inhibitingthe growth of a microbe or the replication of a virus which comprisesthe step of contacting said virus or said microbe with an amount of acompound according to claim 1 effective to inhibit said growth or saidreplication.
 8. The method of claim 7 in which the microbe is abacteria.
 9. The method of claim 7 in which the microbe or virus is asexually-transmitted microbe or virus.
 10. The method of claim 9 inwhich the sexually-transmitted microbe or virus is selected from thegroup consisting of HIV-1, Chlamydia trachomatis, Treponema pallidum,Neisseria gonorrhoeae, Trichonomis vaginalis, HSV-1, HSV-2, Hemophilusducreyi and human papilloma virus
 11. The method of claim 7 in which themicrobe or virus is HIV.
 12. The method of claim 7 in which the microbeor virus is methicillin-resistant S. aureus (MRSA) orvancomycin-resistant E. faecalis (VREF).
 13. A method to treat orprevent a microbial or viral infection in a subject, which methodcomprises administering to a subject in need of such treatment an amountof a compound according to claim 1 effective to ameliorate or preventsaid infection in the subject.
 14. The method of claim 13 in which theinfection is a bacterial infection.
 15. The method of claim 14 in whichthe bacteria is selected from the group consisting of E. Coli, L.monocytogenes, B. subtilis, S. typhimurium, S. aureus and P. aeruginosa.16. The method of claim 13 in which the infection is caused by asexually-transmitted pathogen.
 17. The method of claim 16 in which thesexually-transmitted pathogen is selected from the group consisting ofHIV-1, Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae,Trichonomis vaginalis, HSV-1, HSV-2, Hemophilus ducreyi and humanpapilloma virus.
 18. The method of claim 13 in which the infection is anHIV infection.
 19. The method of claim 13 in which the infection is amethicillin-resistant S. aureus (MRSA) or vancomycin-resistant E.faecalis (VREF) infection.
 20. The method of claim 13 in which thecompound is administered topically.
 21. The method of claim 13 in whichthe compound is administered prophylactically.